Universal sample preparation system and use in an integrated analysis system

ABSTRACT

The invention provides a system that can process a raw biological sample, perform a biochemical reaction and provide an analysis readout. For example, the system can extract DNA from a swab, amplify STR loci from the DNA, and analyze the amplified loci and STR markers in the sample. The system integrates these functions by using microfluidic components to connect what can be macrofluidic functions. In one embodiment the system includes a sample purification module, a reaction module, a post-reaction clean-up module, a capillary electrophoresis module and a computer. In certain embodiments, the system includes a disposable cartridge for performing analyte capture. The cartridge can comprise a fluidic manifold having macrofluidic chambers mated with microfluidic chips that route the liquids between chambers. The system fits within an enclosure of no more than 10 ft 3 . and can be a closed, portable, and/or a battery operated system. The system can be used to go from raw sample to analysis in less than 4 hours.

CROSS-REFERENCE

This application is related to U.S. Ser. No. 61/184,759 filed Jun. 5,2009, U.S. Ser. No. 61/235,664, filed Aug. 20, 2009, U.S. Ser. No.61/349,680 filed May 28, 2010, and PCT/US2010/037545 filed Jun. 4, 2010,which are incorporated herein by reference in their entirety for allpurposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Contract No.2004*H838109*000 awarded by the Central Intelligence Agency. TheGovernment may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Sample preparation is a ubiquitous problem in biological analyticalsystems. The issue of providing sufficiently purified targets fromdiverse raw sample types to reliably perform downstream analyticalassays is pervasive and covers cell biology, genomics, proteomics,metabolomics, food biology, molecular diagnostics, and many otherbiological and medical assays. While many advances in sample preparationhave been made the chief solution has been to develop reagents that areused manually or in robotic systems that use rectilinear stages ormulti-axis arms to manipulate samples.

Microfluidics and nanofluidics allow miniaturized sample volumes to beprepared for analysis. Advantages include the nanoscale consumption ofreagents to reduce operating costs and full automation to eliminateoperator variances. Microfluidic sample preparation can either interfacewith existing or future detection methods or be part of a completelyintegrated system. In the present application, methods and apparatusesare disclosed that integrate full volume sample preparation with volumesover 10 mL with microliter and smaller volumes for sample preparationand analysis.

Starting from the sample, the present invention can be applied toconcentrate, and pre-separate components for further processing todetect and classify organisms in matrices comprising aerosol samples,water, liquids, blood, stools, nasal, buccal and other swabs, bodilyfluids, environmental samples with analysis by ELISA, PCR or othernucleic acid amplification techniques, single molecule detection,protein arrays, mass spectroscopy, and other analytical methods wellknown to one skilled in the art.

Microfluidic nucleic acid purification can be performed to prepare thesample for nucleic acid assays. For DNA analysis, PCR amplification isone current method. Microarray DNA, RNA and protein analysis alsorequires extensive sample preparation before the sample can be appliedto the microarray for reaction and readout.

Samples can be obtained by a wide variety of substrates and matrices.The matrix may contain complex mixtures including inhibitory compoundssuch as hemes, indigo, humic acids, divalent cations, and proteins etcthat interfere with DNA-based amplification. Aerosols can contain largeamounts of molds, metals, and soils humic and other acids that allinterfere with PCR amplification—the gold standard.

Early work showed that as few as three seeded organisms could bedetected from diluted samples of soil extracts followed by PCRamplification of two 16S ribosomal gene fragments.Low-melting-temperature agarose has been used to extract DNA from soilsamples for 16S and 18S rDNA PCR amplification using universal primers.Spun separation gels in column format can be used, such as Sephadexcolumns Multistep purifications such as organic extractions combinedwith Sephadex columns were developed. Bead beating was found to be aneffective way to prepare samples for high numbers of organisms andgrinding in liquid nitrogen to detect low numbers of organisms. Whilethese methods are effective they were best suited for researchlaboratory environments.

Solid phase extractions to columns, beads, and surfaces can be used topurify DNA before DNA analysis. Proteinase K followed by a Qiagen QIAAmp silica-gel membrane columns and IsoCode Stix, an impregnatedmembrane-based technology, followed by heating, washing and a briefcentrifugation were compared for B. anthracis Sterne vegetative cells inbuffer, serum, and whole blood and spores in buffer and found to workwell.

A variety of separations can be performed using the devices and methodsof the invention. For example, the devices and methods of the inventioncan be used to perform chromatography, phase-based or magnetic-basedseparation, electrophoresis, distillation, extraction, and filtration.For example, a microfluidic channel or a capillary can be used forchromatography or electrophoresis. As well, beads, such as magneticbeads can be used for phase-based separations and magnetic-basedseparations. The beads, or any other surfaces described herein, can befunctionalized with binding moieties that exhibit specific ornon-specific binding to a target. The binding can be based onelectrostatics, van der Walls interactions, hydrophobicity,hydrophilicity, hydrogen bonding, ionic interactions, as well aspartially covalent interactions like those exhibited between gold andsulfur. In preferred embodiments, the devices and methods of theinvention utilize immunomagnetic separations.

Immunomagnetic separation (IMS) is a powerful technology that allowstargets to be captured and concentrated in a single step using amechanistically simplified format that employs paramagnetic beads and amagnetic field (see Grodzinski P, Liu R, Yang J, Ward M D. Microfluidicsystem integration in sample preparation microchip-sets—a summary. ConfProc IEEE Eng Med Biol Soc. 2004; 4:2615-8, Peoples M C, Karnes H T.Microfluidic immunoaffinity separations for bioanalysis. J Chromatogr BAnalyt Technol Biomed Life Sci. 2007 Aug. 30., and Stevens K A, Jaykus LA. Bacterial separation and concentration from complex sample matrices:a review. Crit Rev Microbiol. 2004; 30(1):7-24.). IMS can be used tocapture, concentrate, and then purify specific target antigens,proteins, toxins, nucleic acids, cells, and spores. While IMS asoriginally used referred to using an antibody, we generalize its usageto include other specific affinity interactions including lectins,DNA-DNA, DNA-RNA, biotin-streptavidin, and other affinity interactionsthat are coupled to a solid phase. IMS works by binding a specificaffinity reagent, typically an antibody or DNA, to paramagnetic beadswhich are only magnetic in the presence of an external magnetic field.The beads can be added to complex samples such as aerosols, liquids,bodily fluids, or food. After binding of the target to the affinityreagent (which itself is bound to the paramagnetic bead) the bead iscaptured by application of a magnetic field. Unbound or loosely boundmaterial is removed by washing with compatible buffers, which purifiesthe target from other, unwanted materials in the original sample.Because beads are small (about 1 nm to about 1 um) and bind high levelsof target, when the beads are concentrated by magnetic force theytypically form bead beds of between 1 nL and 1 uL, thus concentratingthe target at the same time it is purified. The purified andconcentrated targets can be conveniently transported, denatured, lysedor analyzed while on-bead, or eluted off bead for further samplepreparation, or analysis.

Immunomagnetic separations are widely used for many applicationsincluding the detection of microorganisms in food, bodily fluids, andother matrices. Paramagnetic beads can be mixed and manipulated easily,and are adaptable to microscale and microfluidic applications. Thistechnology provides an excellent solution to themacroscale-to-microscale interface: beads are an almost ideal vehicle topurify samples at the macroscale and then concentrate to the nanoscale(100's of nL) for introduction into microfluidic or nanofluidicplatforms Immunomagnetic separations are commonly used as an upstreampurification step before real-time PCR, electrochemiluminescence, andmagnetic force discrimination.

The ability to move fluids on microchips is a quite important. Thisinvention describes technologies in sample capture and purification,micro-separations, micro-valves, -pumps, and -routers, nanofluidiccontrol, and nano-scale biochemistry. A key component of the technologyis Micro-robotic On-chip Valves (MOVe) technology (an example of whichis shown in FIG. 1) and its application to miniaturize and automatecomplex workflows. Collectively the MOVe valves, pumps, and routers andthe instrumentation to operate them can be referred to as a microchipfluid processing platform.

The heart of the microchip fluid processing platform technology are MOVepumps, valves, and routers that transport, process, and enable analysisof samples. These novel externally actuated, pneumatically-driven,on-chip valves, pumps, and routers, originally developed in the Mathieslaboratory at the University of California at Berkeley (U. C. Berkeley)(Grover, W. H. A. M. Skelley, C. N. Liu, E. T. Lagally, and R. M.Mathies. 2003. Sensors and Actuators B89:315-323; Richard A. Mathies etal., United States Patent Application, 20040209354 A1 Oct. 21, 2004; allof which are herein incorporated by reference in their entirety) cancontrol fluidic flow at manipulate volumes from 20 nL to 10 μL.

The MOVe valves and pumps (FIG. 1) can combine two glass and/or plasticmicrofluidic layers with a polydimethyl siloxane (PDMS) deformablemembrane layer that opens and closes the valve, and a pneumatic layer todeform the membrane and actuate the valve. The microfluidic channeletched in the top glass fluidic wafer is discontinuous and leads to avalve seat which is normally closed (FIG. 1A). When a vacuum is appliedto the pneumatic displacement chamber by conventional-scale vacuum andpressure sources, the normally closed PDMS membrane lifts from the valveseat to open the valve (FIG. 1B). FIG. 1C shows a top view of the valvea similar scale as the other panels.

Three microvalves can be used to make a micropump on a microchip to movefluids from the Input area to the Output area on Microchip A. The fluidsare moved by three or more valves. The valves can be created actuationof a deformable structure. In some implementations a valve seat iscreated and in other embodiments no valve seat may be needed. FIG. 2shows MOVe devices from top to bottom: valve, router, mixer, beadcapture. Self-priming MOVe pumps (FIG. 2, top) are made by coordinatingthe operation of three valves and can create flow in either direction.Routers are made from three or more MOVe valves (FIG. 2, top middlepanel). Mixing has been a holy grail for microfluidics: MOVe mixers(FIG. 2, bottom middle panel) rapidly mix samples and reagents. MOVedevices work exquisitely with magnetic beads to pump or trap sets ofbeads (FIG. 2, bottom panel).

The normally closed MOVe valves, pumps, and routers are durable, easilyfabricated at low cost, can operate in dense arrays, and have low deadvolumes. Arrays of MOVe valves, pumps, and routers are readilyfabricated on microchips. Significantly, all the MOVe valves, pumps, androuters on a microchip are created at the same time in a simplemanufacturing process using a single sheet of PDMS membrane—it costs thesame to make 5 MOVe micropumps on a microchip as to create 500. Thisinnovative technology offers for the first time the ability to createcomplex micro- and nanofluidic circuits on microchips.

Patents and applications which discuss the use and design of microchipsinclude U.S. Pat. No. 7,312,611, issued on Dec. 25, 2007; U.S. Pat. No.6,190,616, issued on Feb. 20, 2001; U.S. Pat. No. 6,423,536, issued onJul. 23, 2002; U.S. Pat. No. 10.633,171 Mar. 22, 2005; U.S. Pat. No.6,870,185, issued on Mar. 22, 2005 US Application No. US 2001-0007641,filed on Jan. 25, 2001; US Application US20020110900, filed on Apr. 18,2002; US patent application 20070248958, filed Sep. 15, 2005; US patentapplication US 20040209354, filed on Dec. 29, 2003; US patentapplication US2006/0073484, filed on Dec. 29, 2003; US20050287572, filedon May 25, 2005; US patent application US20070237686, filed on Mar. 21,2007; US 20050224352 filed on Nov. 24, 2004; US 20070248958, filed on,Sep. 15, 2005; US 20080014576, filed on Feb. 2, 2007; and, USapplication US20070175756, filed on Jul. 26, 2006; all of which areherein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention provides a system that can process a raw biologicalsample, perform a biochemical reaction and provide an analysis readoutin multiplex. For example, the system can extract DNA from a swab,amplify STR loci from the DNA, and analyze the amplified loci and STRmarkers in the sample. The system integrates these functions by usingmicrofluidic components to connect what can be macrofluidic functions.In one embodiment the system includes a sample purification module, areaction module, a post-reaction clean-up module, a capillaryelectrophoresis module and a computer. In certain embodiments, thesystem includes a disposable cartridge for performing analyte capture.The cartridge can comprise a fluidic manifold having macrofluidicchambers mated with microfluidic chips that route the liquids betweenchambers. The system fits within an enclosure of no more than 10 ft³ andcan be a closed, portable, and/or battery operated system. The systemcan be used to go from sample to analysis in less than 4 hours.

In one aspect, this invention provides a system that fits within anenclosure of no more than 10 ft³, the system comprising: (a) a samplepreparation module adapted to capture an analyte from a non-microfluidicvolume on a capture particle and route the captured analyte through amicrofluidic channel; (b) a reaction module comprising a reactionchamber in fluidic communication with the microfluidic channel adaptedto immobilized the captured analyte and perform a biochemical reactionon the analyte in a non-microfluidic volume to produce a reactionproduct; (c) and an analysis module in fluidic communication with thereaction chamber adapted to perform an analysis on the reaction product.In one embodiment, the system is configured to capture the analyte,perform a biochemical reaction on the analyte, and perform an analysison the product in less than 4 hours, in less than 3 hours, or even inless than 2 hours. In one embodiment, the system further comprises adata analysis module configured to receive data about the analysis fromthe analysis module and comprising executable code that transforms thedata and outputs a result of the analysis. In another embodiment, thesystem further comprises a processing module in fluidic communicationwith the reaction chamber and the analysis module and adapted to (1)route the reaction product through a second microfluidic channel into anon-microfluidic processing chamber; (2) process the reaction productand (3) route the processed reaction product into the analysis module.In one particular embodiment, the system fits within an enclosure of nomore than 8 ft³, no more than 5 ft³ or no more than 2½ ft³. In anotherembodiment of the system, the sample preparation module is adapted torelease the analyte from a cell. In another embodiment of the system,the capture particle is a magnetically responsive capture particle and areaction module comprises a source of magnetic force configured toimmobilize the captured analyte. In another embodiment of the system,the reaction module is adapted to perform thermal cycling. In anotherembodiment of the system, the system is a closed system and/or batteryoperated.

In another aspect, this invention provides a system comprising acartridge cover, a cartridge and a pneumatic manifold wherein thecartridge can be releaseably engaged with the cartridge cover and thepneumatic manifold, wherein the cartridge comprises one or morepneumatically actuated valves and one or more microfluidic channels,wherein the pneumatic manifold and the cartridge cover are eachfluidically connected to at least one pressure source, and wherein thepneumatic manifold and the cartridge cover are each adapted to controlfluid flow within the cartridge. In one embodiment, the pneumaticmanifold is adapted to actuate the pneumatically actuated valves and thecartridge cover is adapted to apply pressure to one or more chambers inthe cartridge.

In another aspect, this invention provides a system comprising: (a) adisposable cartridge comprising at least one set of fluidic chambersincluding a sample chamber, a mixing chamber and a thermal cyclingchamber in fluid communication with each other, and a reagent cardcomprising reagents for performing a chemical reaction involving thermalcycling, wherein the reagent card is configured to be carried on thecartridge in a closed configuration and to be moved into fluidcommunication with the at least one set of fluidic chambers; (b) anactuator assembly configured to move fluids between chambers when thecartridge is engaged with the actuator assembly; (c) a thermal cyclerconfigured to cycle temperature in the thermal cycling chamber when thecartridge is engaged with the actuator assembly; (d) a capillaryelectrophoresis assembly configured to accept a sample from cartridgewhen the cartridge is engaged with the actuator assembly and to performcapillary electrophoresis on the sample; and (e) a computerized controlsystem configured to control the actuator assembly, the thermal cyclerand the capillary electrophoresis assembly.

In another aspect, this invention provides a cartridge comprising: (a) afluidic manifold comprising a fluidic side and a reagent card sidewherein the fluidic manifold comprises: (i) at least one set of fluidicchambers, each chamber comprising a port on the fluidic side; (ii) atleast one of thermal cycling chamber comprising at least one port; (iii)at least one of exit port; (iv) a slot on the reagent card side adaptedto engage a reagent card, wherein the slot comprises a plurality of slotchannels comprising cannulae on the reagent card side and communicatingbetween the two sides; (b) at least one microfluidic chip comprising: atleast one fluidic circuit; (ii) a plurality of ports in fluidcommunication with the fluidic circuit; (iii) at least one pneumaticallyactivated diaphragm valve configured to regulate fluid flow within thefluidic circuit; wherein the at least one chip is engaged with thefluidic manifold so that the ports in the at least one chip are in fluidcommunication with the ports of the chambers and the slot channelswherein each fluidic chamber is in fluid communication with at least oneother fluidic chamber and each cannula is in communication with afluidic chamber; and (c) a reagent card engaged with the slot, whereinthe card comprises a plurality of reagent chambers comprising reagents,each aligned with at least one of the cannulae and adapted to take afirst engagement position wherein the reagent chambers are not puncturedby the cannulae and a second engagement position wherein the reagentchambers are punctured by the cannulae, thereby putting the reagentchambers in fluid communication with the fluidic circuit. In oneembodiment the reagents comprise reagents for performing PCR. In anotherembodiment, the reagents comprise primers for amplifying a plurality ofshort tandem repeats. In another embodiment, the at least one set offluidic chambers is a plurality of sets of fluidic chambers. In yetanother embodiment the cartridge is such that the fluidics manifoldfurther comprises at least one auxiliary fluidic channel on the fluidicside of the manifold, the at least one chip is a plurality of chips andfluidic circuits in each of the plurality of chips are in fluidiccommunication with fluidic circuits of at least one other chip throughthe auxiliary fluidic channel. In this embodiment, the cartridge canfurther comprise a gasket between the chips and the manifold, whereinthe gasket seals the channels on the manifold. In another embodiment ofthe cartridge, the fluidic chambers comprise a distribution chamber, acapture chamber, a sample chamber, and a clean-up chamber. In anotherembodiment of the cartridge, at least one fluidic chamber comprisesmagnetically responsive particles. In another embodiment of thecartridge, the at least one set of fluidic chambers is at least 4 setsor at least 8 sets. In another embodiment of the cartridge, the chipscomprise at least one diaphragm valve.

In another aspect, the invention provides a system comprising: (a) apneumatic assembly comprising: (i) a pneumatic manifold adapted toremovably engage the cartridge on the fluidic side, wherein thepneumatic manifold comprises a plurality of pneumatic ports configuredto engage pneumatic channels in the at least one microfluidic chip andactivate the diaphragm valves; and (ii) a pressure source configured tosupply positive or negative pressure to the pneumatic channels; (b) acartridge activation assembly adapted to engage the cartridge on thereagent card side; wherein the cartridge activation assembly comprises:(i) a reagent pneumatic manifold comprising a pneumatic side and reagentcard side, wherein the reagent pneumatic manifold comprises reagentpneumatic manifold channels communicating between the two sides andcomprising a cannula on the reagent card side; (ii) a pressure sourceconfigured to supply positive or negative pressure to the reagentpneumatic manifold channels; and (iii) a clamp configured to move thereagent card from the first engagement position to the second engagementposition, wherein clamping results in the cannulae of the reagentpneumatic manifold puncturing the reagent chambers and putting thereagent chambers in communication with the pressure source; (c) athermal cycler configured to cycle temperature in the at least onethermal cycling chamber when the cartridge is clamped; (d) a capillaryelectrophoresis assembly comprising: (i) at least one separation channelfluidically engaged with the exit port when the cartridge is clamped;and (ii) an optical sub-assembly configured to detect signal from the atleast one separation channel; and (e) a computerized control systemconfigured to control pneumatic assembly, the cartridge activationassembly, the thermal cycler and the capillary electrophoresis assembly.In one embodiment of this system, clamping seals the chamber ports andthe slot channels with the at least one microfluidic chip. In anotherembodiment of this system, the cartridge activation assembly furthercomprises at least one heater configured to heat at least one of thefluidic chambers when the reagent pneumatic manifold is engaged with thecartridge. In another embodiment of this system, the cartridgeactivation assembly further comprises movable magnets configured to moveinto and out of a position wherein the magnets exert a magnetic force onat least one fluidic chamber. In another embodiment of this system, thecartridge activation assembly further comprises sensors configured todetect the presence of a sample in a sample chamber of the fluidicmanifold. In another embodiment of this system, the thermal cyclercomprises a Peltier device. In one embodiment the system is comprised ina portable case. In one specific embodiment, the case has an internalvolume of no more than 10 ft³ or no more than 2½ ft³. In a relatedembodiment of system, the invention provides for an article in computerreadable form comprising code for the operating system.

In another aspect the invention provides a method comprising: producing,from a sample comprising at lest one cell comprising DNA, a computerfile identifying a plurality of STR markers in the DNA, wherein themethod is performed in less than 4 hours. In one embodiment the methodis performed in less than 3 hours. In another embodiment the method isperformed in less than 2 hours. In a related embodiment, the producingcomprises extracting the DNA from the at least one cell, amplifying theSTR markers from the DNA, performing capillary electrophoresis on theamplified markers, detecting the amplified markers, and performingcomputer analysis on the detected amplified markers to identify themarkers. In one embodiment, the plurality of STR markers is at least 5STR markers. In another embodiment the plurality of markers are CODISSTR markers. In a related embodiment, the plurality of STR markers is atleast 5, 10, or 13 CODIS STR markers. In these embodiments, the at leastone cell can be a plurality of cells. In some embodiments, the sample isa forensic sample. In specific embodiments, the method is performed atthe site of a sample collection. The sample can comprise blood, or cancomprise a cheek swab. The method can be carried out by any systemdescribed herein.

In a related aspect, the invention provides a system configured toperform a method, wherein the method comprises: producing, from a samplecomprising at least one cell comprising DNA, a computer file identifyinga plurality of STR markers in the DNA, wherein the method is performedin less than 4 hours.

In another aspect, this invention provides a method comprising: (a)providing a system comprising: (i) a disposable cartridge comprising atleast one set of fluidic chambers including a sample chamber, a mixingchamber and a thermal cycling chamber in fluid communication with eachother, and a reagent card comprising reagents for performing a chemicalreaction involving thermal cycling, wherein the reagent card isconfigured to be carried on the cartridge in a closed configuration andto be moved into fluid communication with the at least one set offluidic chambers; (ii) an actuator assembly configured to move fluidsbetween chambers when the cartridge is engaged with the actuatorassembly; (iii) a thermal cycler configured to cycle temperature in thethermal cycling chamber when the cartridge is engaged with the actuatorassembly; (iv) a capillary electrophoresis assembly configured to accepta sample from cartridge when the cartridge is engaged with the actuatorassembly and to perform capillary electrophoresis on the sample; and (v)a computerized control system configured to control the actuatorassembly, the thermal cycler and the capillary electrophoresis assembly;(b) moving of the reagent card into fluid communication with at leastone set of fluidic chambers; (c) providing a sample comprising a nucleicacid molecule to a sample chamber; and (d) operating the system toamplify and detect at least one nucleic acid sequence in the sample. Inone embodiment the time it takes to go from step (b) to step (d) is lessthan 4 hours. In one embodiment, the method comprises providing each ofa plurality of samples to a different sample chamber. In anotherembodiment, the method comprises amplifying and detecting a plurality ofnucleic acid sequences in the sample. In a related embodiment, theplurality of nucleic acid sequences comprise short tandem repeats(STRs). In a specific embodiment, the short tandem repeats comprise aplurality of Combined DNA Index System (CODIS) markers. In anotherembodiment the CODIS markers comprise a plurality of markers selectedfrom AMEL, D3S1358, THO1, D21s11, D18s51, D5s818, D13s317, D7s820,D16s539, CSF1PO, vWA, D8S1179, TPOX and FGA. In one embodiment, thesystem comprises: (a) a pneumatic assembly comprising: (i) a pneumaticmanifold adapted to removably engage the cartridge on the fluidic side,wherein the pneumatic manifold comprises a plurality of pneumatic portsconfigured to engage pneumatic channels in the at least one microfluidicchip and activate the diaphragm valves; and (ii) a pressure sourceconfigured to supply positive or negative pressure to the pneumaticchannels; (b) a cartridge activation assembly adapted to engage thecartridge on the reagent card side; wherein the cartridge activationassembly comprises: (i) a reagent pneumatic manifold comprising apneumatic side and reagent card side, wherein the reagent pneumaticmanifold comprises reagent pneumatic manifold channels communicatingbetween the two sides and comprising a cannula on the reagent card side;(ii) a pressure source configured to supply positive or negativepressure to the reagent pneumatic manifold channels; and (iii) a clampconfigured to move the reagent card from the first engagement positionto the second engagement position, wherein clamping results in thecannulae of the reagent pneumatic manifold puncturing the reagentchambers and putting the reagent chambers in communication with thepressure source; (c) a thermal cycler configured to cycle temperature inthe at least one thermal cycling chamber when the cartridge is clamped;(d) a capillary electrophoresis assembly comprising: (i) at least oneseparation channel fluidically engaged with the exit port when thecartridge is clamped; and (ii) an optical sub-assembly configured todetect signal from the at least one separation channel; and (e) acomputerized control system configured to control pneumatic assembly,the cartridge activation assembly, the thermal cycler and the capillaryelectrophoresis assembly. In another embodiment, the system is anysystem named herein. In one embodiment, the sample is a forensic sample.In a related embodiment, the sample is selected from a buccal swab,blood, hair or semen. In another embodiment, the sample is a raw sample.In some embodiments, the method further comprises transporting thesystem to a forensic site.

In another aspect, the invention provides an optical system comprising:(a) a plurality of optically transparent channels; (b) a light sourceconfigured to direct to the plurality of optically transparent channels;(c) a dispersive element that disperses light passing through theoptically transparent channels in a wavelength dependent manner; and (d)a detector configured to receive the dispersed light. In one embodimentthe plurality of optically transparent channels comprises at least eightcapillaries. In another embodiment the optically transparent channelsare aligned in a first plane and the dispersive element disperses lightalong a second plane, wherein the first plane and the second plane aredifferent. In another embodiment, the first plane is orthogonal to thesecond plane.

In another aspect, the invention provides an optical system comprising:(a) an excitation source configured to direct excitation light to anobject; (b) a carrier for an object, wherein the object emits lightother than excitation light when excited by the excitation energy; (c) arejection filter configured to filter out excitation energy and to allowtransmission of the emitted light; (d) an imaging lens configured tofocus the emitted light; (e) a dichroic mirror substantially transparentto the excitation energy and configured to reflect emitted light to adetector; (f) a focusing system comprising at least one lens configuredto focus light reflected from the dichroic mirror; and (g) aphotodetector (CCD camera) configured to receive the reflected light. Inone embodiment of the optical system, the excitation light compriseslight of a wavelength between 03 microns and 1 micron. In anotherembodiment, the carrier comprises an array of capillary tubes and theobject comprises a fluorescent species. In another embodiment, themirror reflects emitted light and an angle between about 5 degrees andabout 10 degrees off an incident angle. In another embodiment, thedichroic mirror further comprises a portion that transmits substantiallyall light. In another embodiment, the focusing system comprises at leastone folding mirror. In another embodiment, the photodetector comprises aCCD camera. In a related embodiment, the optical system can furthercomprise a prism located between the object and the imaging lens.

In another aspect, the invention provides an optical system comprising:(a) an array of capillary tubes aligned substantially parallel andsubstantially in a plane; (b) an excitation assembly comprising anexcitation source and configured to deliver excitation light from theexcitation source to the array, wherein the light delivery assembly isconfigured (i) to deliver a thin band of light that covers the array and(ii) to deliver the light to the array at an angle other than 90 degreesto the plane; (c) a collection lens configured to collect light emittedfrom the array by objects in the array excited by the excitation light;wherein the excitation assembly and the collection lens are configuredwith respect to the array so that excitation light passing through thearray substantially avoids collection by the collection lens. In oneembodiment, the angle is between about 10 degrees and about 85 degrees.

In another aspect, the invention provides an instrument comprising: (a)a microfluidic component comprising a plurality of intersectingmicrofluidic channels and at least one controllable valve configured toregulate flow of fluid between the intersecting channels; and (b) anon-microfluidic component comprising a plurality of non-microfluidicchambers, wherein each non-microfluidic chamber is fluidically connectedto at least one of the microfluidic channels; wherein the instrument isconfigured to flow fluid from at least one non-microfluidic chamber intoanother non-microfluidic chamber through a microfluidic channel and flowis regulated by at least one valve. In one embodiment of the instrument,the plurality of non-microfluidic chambers comprises at least threechambers and the at least one valve selectively directs fluid from onechamber to either of the at least two other chambers. In anotherembodiment, the instrument further comprises a pump to pump fluid from anon-microfluidic chamber into a microfluidic channel. In anotherembodiment of the instrument the at least one valve is a diaphragmvalve. In a related embodiment, the pump is a diaphragm pump comprisinga series of three diaphragm valves. In another embodiment, themicrofluidic component comprises a monolithic device. IN anotherembodiment, the combination of the microfluidic component and thenon-microfluidic component define a fluidic circuit and the instrumentcomprises a plurality of fluidic circuits. In another embodiment, thenon-microfluidic component further comprises a particulate captureagent. In another embodiment, the particles are responsive to a magneticfield and the instrument further comprises a magnet configured toimmobilize the particles. In another embodiment, the invention providesa device comprising a plurality of non-microfluidic chambers fluidicallyconnected to a common microfluidic channel.

In another aspect, the invention provides a method comprising: (a)providing a device comprising: (i) a microfluidic component comprising aplurality of intersecting microfluidic channels and at least onecontrollable valve configured to regulate flow of fluid between theintersecting channels; and (ii) a non-microfluidic component comprisinga plurality of non-microfluidic chambers, wherein each non-microfluidicchamber is fluidically connected to at least one of the microfluidicchannels; wherein the instrument is configured to flow fluid from atleast one non-microfluidic chamber into another non-microfluidic chamberthrough a microfluidic channel and flow is regulated by at least onevalve; and wherein a first non-microfluidic chamber comprises a firstvolume of sample comprising an analyte; (b) providing an amount ofparticulate capture agent in the first non-microfluidic chamber to binda selected amount of analyte from the sample; (c) moving the particulatecapture agent bound to the analyte through a microfluidic channel in themicrofluidic device to a second non-microfluidic chamber; (d) contactingthe particulate capture agent bound to the analyte with a reagent in asecond non-microfluidic chamber; and (e) performing a chemical reactionon the analyte using the reagent. In one embodiment of the method thecontacting comprises flowing the reagent from a third non-microfluidicchamber through a microfluidic channel in the microfluidic device intothe second non-microfluidic chamber. In another embodiment of themethod, the particles are response to magnetic force and the methodfurther comprises immobilizing the particulate capture agent bound tothe analyte in the instrument with a magnetic force. In anotherembodiment, the method comprises suspending the particulate captureagent bound to the analyte in the instrument in a volume at least anorder of magnitude smaller than the sample volume.

In another aspect, the invention provides a method comprising: (a)performing a first chemical reaction on an analyte in a first chamberwhich is a non-microfluidic chamber to produce a first reaction product;and (b) moving the first reaction product through a microfluidic channelinto a second chamber which is a non-microfluidic chamber and performinga second chemical reaction on the first product to create a secondreaction product.

In another aspect, the invention provides a method comprising: (a)performing a first chemical reaction on an analyte in a first chamberwhich is a non-microfluidic chamber to produce a first reaction product;and (b) moving the first reaction product through a microfluidic channelinto a second chamber which is a microfluidic chamber and performing asecond chemical reaction on the first product to create a secondreaction product. In a related aspect of the invention, provided hereinis a method comprising: (a) performing a first chemical reaction on ananalyte in a first chamber which is a microfluidic chamber to produce afirst reaction product; and (b) moving the first reaction productthrough a microfluidic channel into a second chamber which is anon-microfluidic chamber and performing a second chemical reaction onthe first product to create a second reaction product. In oneembodiment, the methods of these related aspects comprise cleaning thefirst reaction product before moving it to the second chamber. Inanother embodiment, the methods of these related aspects comprise atleast once, moving a reaction product through a microfluidic channelinto a subsequent non-microfluidic chamber and performing a subsequentchemical reaction on the reaction product to create a subsequentreaction product. In another embodiment, the methods of these relatedaspects comprise at least once and before moving a reaction product intoa non-microfluidic chamber, moving a reaction product through amicrofluidic channel into a microfluidic chamber and performing asubsequent chemical reaction on the reaction product to create asubsequent reaction product.

In another aspect of the invention, provided herein is a devicecomprising: (a) a sample channel having a channel inlet and a channeloutlet; (b) an electrophoresis capillary having a capillary inlet and acapillary outlet, wherein the capillary comprises an electricallyconductive medium and is in communication with the sample channel at apoint of connection; (c) an anode and a cathode configured to apply avoltage across the capillary inlet and capillary outlet, wherein one ofthe anode or cathode comprises a forked electrode wherein the forks arein electrical communication with the sample channel on different sidesof the point of connection; and (d) a second electrode in electricalcommunication with the sample channel substantially opposite the pointof connection. In one embodiment of the device, the second electrode iscomprised as a third fork in the forked electrode.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts an example of a microscale on-chip valve (MOVe).

FIG. 2 shows a MOVe microvalve, a microrouter, a MOVe mixer, and beadcapture on microchips.

FIG. 3 shows a fluidic cartridge with MOVe microvalves.

FIG. 4 shows a fluidic cartridge with ports to a microfluidic microchipwith microvalves.

FIG. 5 shows a microfluidic microchip with MOVe valves that controlsflows in a cartridge.

FIG. 6 shows a cartridge connected to reaction chamber and detector withdownstream MOVe pumps and reagents.

FIG. 7 shows a temperature control device that can thermal cycle andincorporates magnetic capture, pinch clamps and the capability ofcycling seven reactions simultaneously.

FIG. 8 shows a temperature control device that can thermal cycle andincorporates magnetic capture, pinch clamps and the capability ofcycling seven reactions simultaneously.

FIG. 9 shows PowerPlex16 STR (Short tandem repeat) amplificationreaction performed in a passive, Teflon (PTFE) based Tube reactionchamber.

FIG. 10 shows purification of DNA from 25 uL of blood at 69′, 23.5′,10.5′, and 4.5′; yield in ng is shown on the bars.

FIG. 11 shows a schematic of using microvalves to capture beads on amicrochip.

FIG. 12 shows bead capture from a cartridge on a microchip using a MOVemicrovalve.

FIG. 13 shows bead capture from a cartridge on a microchip using a MOVemicrovalve.

FIG. 14 shows a capture and reaction microchip using MOVe microvalves.

FIG. 15 shows a capture and reaction microchip using MOVe microvalves.

FIG. 16 shows a four module assembly.

FIG. 17 shows an example of STR reactions on microchips.

FIG. 18 shows a universal sample preparation workflow to prepare nucleicacids and toxins.

FIG. 19 shows purification of samples in a cartridge using paramagneticbeads.

FIG. 20 shows an integrated pneumatic manifold to operate the MOVemicrovalves in cartridge.

FIG. 21 shows a cartridge mounted on a computer controlled apparatus.

FIG. 22 shows a cartridge mounted on a computer controlled apparatus.

FIG. 23 shows a reagent distribution manifold based on MOVe technologythat can distribute five reagents to five extraction/isolation or otherdevices.

FIG. 24 shows a reagent distribution manifold based on MOVe technologythat can distribute five reagents to five extraction/isolation or otherdevices.

FIG. 25 shows a distribution manifold with sample loops and MOVemicrovalves.

FIG. 26 shows a pneumatic manifold, top panel shows the top side and thelower panel the bottom side.

FIG. 27 shows detection of E. coli by immunomagnetic separation,followed by alkaline lysis and PEG-facilitated capture on magneticbeads, and analyzed by real-time PCR.

FIG. 28 shows application of a cartridge with three chambers that can beused to construct genomic libraries and other applications.

FIG. 29 shows the workflow to prepare genomic libraries using thecartridge.

FIG. 30 shows a forked injector for microchip based electrophoresis.

FIG. 31 shows sample stacking with a forked injector.

FIG. 32 shows a forked injector coupled to MOVe microvalves.

FIG. 33 shows a forked cathode injector coupled with a MOVe microchip.

FIG. 34 shows a photograph of a microchip with the forked injector.

FIG. 35 shows a photograph of a microchip with the forked injector.

FIG. 36 shows an electropherogram of a single color from a DNAsequencing trace from a forked cathode injector.

FIG. 37 shows STR separations on a forked cathode injection system.

FIG. 38 shows a forked cathode with MOVe microfluidics for shuttleloading.

FIG. 39 shows an integrated system for nucleic acid isolation,amplification(s), separation and detection.

FIG. 40 depicts a device with a cartridge, microfluidic microchip, and amagnet.

FIG. 41 depicts a microfluidic microchip with a fluidics layer, anelastomeric layer, and a pneumatics layer.

FIG. 42 depicts a fluidics layer made of two layers of material.

FIG. 43 depicts a fluidics layer made of a single layer of material.

FIG. 44 depicts a reaction scheme for amplifying mRNA.

FIG. 45 shows a microfluidic microchip with MOVe valves that controlsflows in a cartridge.

FIG. 46 shows a forked electrode.

FIG. 47 shows a forked electrode, a forked electrode with a wire runelectrode, and a forked electrode with a cannular electrode.

FIG. 48 shows sample injection into a separation channel.

FIG. 49 shows a device for mating a separation capillary with injectiontubing.

FIG. 50 shows a device for mating separation capillaries with fourinjection tubings.

FIG. 51 shows a thermocycler with an Ultem pinch clamp.

FIG. 52 shows a diagram indicating movement of reagents betweencomponents of a four channel parallel processing device.

FIG. 53 shows a four-channel parallel reagent delivery device: the ChipC microchip design is shown on the top left, a fluidic manifold is shownon the bottom left, and the fabricated and assembled device is shown onthe right.

FIG. 54 shows a four-channel sample preparation device on the left and afour-channel sample preparation device mounted on a monolithic pneumaticmanifold on the right.

FIG. 55 shows MOVe microchip designs of the four-channel samplepreparation device.

FIG. 56 shows IdentiFiler STR profiles of DNA samples prepared on thefour-channel sample preparation device, where STR amplifications wereperformed using fast protocols (1.5 hrs) on a STR Reaction subsystemthermocycler.

FIG. 57 shows a four-channel post amplification device combined with anChip A microchip with a fluidics manifold: the Chip A microchip designis shown on the left, the fabricated microchip is shown in the center,and the assembled fluidic manifold and microchip is shown on the right.

FIG. 58 shows a post-amplification STR clean-up subsystem with thepost-amplification device.

FIG. 59 shows the Chip E microchip design, which can be used in thepost-amplification device.

FIG. 60 shows a diagram of a mixer.

FIG. 61 shows a diagram of a mixer.

FIG. 62 shows results of using a mixer to lyse cells.

FIG. 63 depicts components of an integrated analysis system.

FIG. 64 depicts hardware components of an integrated analysis system.

FIG. 65 depicts software components of an integrated analysis system.

FIG. 66 depicts consumable components of an integrated analysis system.

FIG. 67 depicts documentation components of an integrated analysissystem.

FIG. 68 shows a picture of an encased integrated analysis system.

FIG. 69 shows a schematic of an encased integrated analysis system.

FIG. 70 shows a schematic of an encased and portable integrated analysissystem.

FIG. 71 shows a schematic of an encased and portable integrated analysissystem and a polymer injection system.

FIG. 72 shows a schematic of an optical detection system and pneumaticsystem.

FIG. 73 shows a diagram of an integrated analysis system.

FIG. 74 shows a schematic of a cartridge cover and pneumatic components.

FIG. 75 shows a schematic of a cartridge cover, cartridge, pneumaticmanifold, and thermocycler.

FIG. 76 depicts a cartridge with reagent cassette.

FIG. 77 depicts a cartridge with reagent cassette.

FIG. 78 depicts a top view of a cartridge with reagent cassette.

FIG. 79 depicts a top view of a cartridge without reagent cassette.

FIG. 80 depicts a bottom view of a cartridge.

FIG. 81 depicts a bottom view of a cartridge with tubes.

FIG. 82 depicts a bottom view of a cartridge with tubes.

FIG. 83 depicts loading of a cartridge into an encased integratedanalysis system.

FIG. 84 depicts loading of a cartridge into an encased integratedanalysis system.

FIG. 85 depicts loading of a cartridge into an encased integratedanalysis system.

FIG. 86 shows a diagram of a sample analysis procedure.

FIG. 87 shows a diagram of a sample analysis procedure.

FIG. 88A shows a timeline of a sample analysis procedure.

FIG. 88B shows a timeline of a sample analysis procedure.

FIG. 88C shows a timeline of a sample analysis procedure.

FIG. 89 shows a platform that carries an array of microcapillaries andthat comprises an aperture that limits the amount of stray lightcollected by the optics.

FIG. 90 shows an optical assembly.

FIG. 91 shows transmission of light through a plurality of capillaries,each forming a separate image.

FIG. 92 shows an optical assembly comprising a dichroic mirror (foldingmirror 1) that allows transmission of light from the excitation sourceand a second folding mirror that decreases the footprint of theassembly.

FIG. 93 shows the direction of excitation light at an oblique angle tothe plane on which an array of capillaries is arranged, such that lightpassing through the capillaries is not directed normal to the planewhere emitted fluorescent light can be collected.

FIG. 94 is a top-down view of the configuration of FIG. 93.

FIG. 95 shows an image produced by eight capillary tubes that carry afluorescent species and that have been excited by light.

FIG. 96 shows an injection system for injecting a sample into anelectrophoresis capillary.

FIG. 96A shows three electrodes opposite a capillary inside the lumen oftubing. The third electrode is not electrically connected, but isindependently electrified.

FIG. 96B shows three electrodes opposite a capillary inside the lumen oftubing. All the electrodes are electrically connected.

FIG. 97 shows integrated subsystems.

FIGS. 98A and B show embodiments of a device for regulating temperatureof electrophoresis capillaries, for example an array of capillaries. Theelectrically insulating circuit board has a generally S-shaped path forplacement of capillaries.

FIG. 99 shows one embodiment of the optical system for detectinganalytes in an array of capillaries.

FIG. 100 shows ‘precious reagent’ delivery one embodiment of theplacement of ‘precious reagent cartridges’ in the device.

FIG. 101 shows the fluidics and pneumatics architecture of amicrofluidic chip which is then mated with a fluidics cartridge to formthe post amplification module.

FIG. 102 shows one embodiment of the system enclosure which measure2×2×2 ft.

FIG. 103 shows the fluidics and pneumatics architecture of amicrofluidic chip which is then mated with a fluidics cartridge to formthe reagent delivery module.

FIG. 104 shows a portion of a device in which the fluidics layercomprises a plurality of sublayers, in an exploded view. The top orexternal sublayer 121 is referred to as the “etch” layer and bottom orlower sublayer 122 is referred to as the “via” layer. Fluid traveling ina channel in the etch layer can flow into a conduit in the via layerthat faces the elastic layer.

DETAILED DESCRIPTION OF THE INVENTION

This invention includes devices that incorporate valves, such asmicrovalves (including but not limited to pneumatically actuated valvesand microscale on-chip valves), into their design in order to controlthe movement of fluid. These devices can be used for the enrichment of acomponent, for sample preparation, and/or for analysis of one or morecomponents in or from a sample.

The invention also provides devices for fluid and analyte processing andmethods of use thereof. The devices of the invention can be used toperform a variety of actions on the fluid and analyte. These actions caninclude moving, mixing, separating, heating, cooling, and analyzing. Thedevices can include multiple components, such as a cartridge, amicrofluidic microchip, and a pneumatic manifold.

Microfluidic-Non-Microfluidic Integration

In one aspect, this invention provides an instrument that usesmicrofluidic components to integrate functions performed on anon-microfluidic scale. The instrument includes non-microfluidicchambers fluidically connected to each other and to microfluidicchambers through microfluidic channels in a microfluidic device, e.g., amicrofluidic chip. The microfluidic device comprises directional controlmechanisms, such as valves and pumps, by which fluid can be selectivelyrouted between different chambers and along different channels, and bywhich a single chamber can communicate with a number of other chambers.These connections and routing mechanisms allow automation of thefunctions. In certain embodiments, the instrument uses particulatecapture agents that can bind an analyte, be immobilized, be suspended orre-suspended in a desired volume of fluid and be routed to and fromchambers through the microfluidic channels. This simplifies in thetransition of an analyte from a non-microfluidic environment to amicrofluidic environment and back to a non-microfluidic environment. Theamount of capture agent can be selected to capture a desired amountanalyte from a sample, for example, to concentrate analyte from a dilutesample, to quantitatively capture all or substantially all of an analytefrom a sample or to select a portion of an analyte from a moreconcentrated sample. This allows one to “tune” the amount of analyteused in a reaction and further decreases fluid handling steps. Theinstrument can comprise a plurality of parallel fluidic circuits bywhich fluidic operations can be performed in parallel. The integrationof microfluidic and non-microfluidic components on the device simplifiessample handling during the performance of chemical reactions (e.g.,chemical reaction, biochemical reaction and enzymatic reaction). It alsoallows size reduction of operations, decreasing the footprint and thetotal space occupied by the system. Accordingly, the instruments of thisinvention integrate non-microfluidic and microfluidic environments.

More specifically, this invention provides instruments that comprise amicrofluidic component and a non-microfluidic component in fluidcommunication with each other. A microfluidic component comprises atleast one microfluidic channel. A microfluidic channel generally has across sectional area of less than 1 mm² or at least one cross-sectionaldimension in the range of from about 0.1 microns to about 500 microns,e.g., below about 350 microns. A non-microfluidic component comprises atleast one non-microfluidic chamber (open or closed), that is, a chamberthat holds a non-microfluidic volume e.g., at least 10 microliters, atleast 20 microliters, at least 50 microliters, at least 100 microliters,at least 500 microliters, at least 1 ml, at least 10 ml or at least 100ml. Accordingly, in the instruments of this invention, anon-microfluidic chamber is in fluid communication with a microfluidicchannel. The instruments are adapted to transport fluid samples (e.g., aliquid) from a non-microfluidic chamber into a microfluidic channeland/or from a microfluidic channel into a non-microfluidic chamber orotherwise out of the microfluidic device. In other embodiments themicrofluidic devices comprise microfluidic chambers in fluidcommunication with microfluidic channels. In this case, the instrumentsare adapted to transport fluid samples between microfluidic chambers andnon-microfluidic chambers through microfluidic channels. Fluid movedfrom a microfluidic channel into a non-microfluidic chamber can beremoved from the device.

Manipulation of the analyte can include a variety of steps. These caninclude, for example, preparing the analyte for a chemical reaction,mixing with one or more reagents in various sequences, performing achemical reaction with the analyte, removing wastes and washing. Theinstrument of this invention can be used to perform these functions byrouting analytes, reagents, wastes and wash solutions betweencompartments. Accordingly, in certain embodiments the instruments ofthis device comprise a plurality of non-microfluidic chambers connectedto each other through microfluidic channels. Fluid can be moved from onechamber to another by any appropriate motive force, for example,continuous pressure or non-continuous pressure (e.g., positivedisplacement pumps), electroosmotic flow or centrifugation. Pressure canbe generated internally (e.g., with on-chip diaphragm valves) orexternally. The channels can comprise directional control mechanisms toselectively route fluids between chambers as desired. These mechanismscan be valves, such as the diaphragm valves described elsewhere in thisspecification, single use valves such as wax valves or other valves. Byopening and closing valves in a proper sequence, analyte, reagents andwaste can be routed into appropriate locations. In this way, themicrofluidic portion of the instrument is used to route fluids betweennon-microfluidic environments where various functions can be performedon the analyte.

The non-microfluidic chambers can comprise capture agents to bindanalytes. The capture agents generally comprise a solid substrate andare able to specifically or non-specifically bind analytes. Thesubstrate can assert the binding force, or a molecule having bindingproperties can be attached to the substrate, for example, an antibody.The capture agent can be a particulate capture agent. Alternatively, thematerial can be a chromatographic material. In this case, a sample canbe passed through the chromatographic material and separated fractionsintroduced into the microfluidic device. Alternatively, the captureagent can be a monolith. Alternatively, the capture agent can beattached to a surface of the chamber, such as a post, or the chambersurface can be derivatized with a capture molecule.

The device is further adapted to move particles, such as beads, betweena microfluidic channel and a non-microfluidic chamber. The particles canbe responsive to magnetic force, electrical forces, or other forces. Forexample, they can be paramagnetic or magnetic particles. This allowsfurther manipulation of the particles within the device by applicationof magnetic fields using fixed or movable magnets includingelectromagnets. The particles can function as a capture agent to captureone or more analytes from a sample. For example, the particles can havespecific or non-specific affinity for an analyte. Because the particlesare solid, they allow one to transit between non-microfluidic fluidvolumes and microfluidic fluid volumes by immobilizing the particles andaltering the volume of the fluid in which they are contained. Particlescan be immobilized in the non-microfluidic chamber or in themicrofluidic device.

The instrument of this invention can be used to capture a selectedamount of an analyte from a sample in a non-microfluidic chamber andtransport that amount of analyte from the chamber into a microfluidicchannel. Once in the microfluidic channel, the particles can be routedinto a non-microfluidic chamber for storage or processing. For example,a non-microfluidic volume of a sample is provided to a chamber. Thesample may be, for example, dilute or concentrated with respect to ananalyte. By properly calibrating the amount of capture agent andconditions, a selected amount of the analyte can be captured by thecapture agent. For example, in a concentrated sample, a small amount ofcapture agent can be selected so that only a portion of the analyte tobe used in a reaction is captured. This allows sampling of a portion ofthe analyte in the sample volume, e.g., an amount sufficient orappropriate to perform a chemical reaction. Alternatively, in a dilutesample, a greater amount of capture agent can be used so that most orsubstantially all of the analyte present is captured on the captureagent. This effectively concentrates the analyte. Washing the analytecan remove impurities, effectively purifying the analyte fromundesirable components of the sample such as inhibitors, other analytes,etc. The specificity of the capture can also be controlled by adjustingthe chemistry to select broader or narrower ranges of analytes, forexample longer or short pieces of DNA, differentiating DNA from RNA, orusing affinity reagents with broader or narrower specificity. In someembodiments, multiple analytes may be captured by combining affinityreagents or by using multiple modes of capture either on one type ofbead or by mixing multiple bead or particle types. In certainembodiments, the instrument further comprises a detector, e.g., anoptical detector, for detecting molecules at some stage of the process.

A variety of reaction sequences are contemplated. A chemical reactioncan be performed on an analyte. Then, the reaction product can becleaned and subjected to a different chemical reaction. This sequence ofcleaning a reaction product and performing a subsequent differentreaction can be repeated. In particular, this invention contemplatesperforming series of biochemical or enzymatic reactions separated byclean-up steps. One example of this is Eberwine chemistry, in which afirst chemical reaction, reverse transcription, is followed by a secondreaction, second strand synthesis, which is followed by a thirdreaction, transcription. Before each subsequent reaction, and typicallyafter the last reaction, the product is removed from contaminants inpreparation of the next step. In one embodiment, a reaction product in anon-microfluidic chamber is moved through a microfluidic channel toanother non-microfluidic chamber. In another embodiment, a reactionproduct in a non-microfluidic chamber is moved through a microfluidicchannel into a microfluidic chamber, e.g., on-chip. In anotherembodiment, a reaction product in a microfluidic chamber is movedthrough a microfluidic channel into a non-microfluidic chamber.Accordingly, this invention provides the use of small analyte volumesand small reagent volumes, thereby reducing waste of reagent.

For example, the instrument can be used to perform chemical reactions onnucleic acids, such as DNA amplification. A non-microfluidic volume of asample (e.g., several hundred microliters) comprising DNA can becollected in a non-microfluidic chamber of this device. Paramagneticcapture particles can be flowed through microfluidic circuits into thechamber to capture a calibrated amount of nucleic acid. The paramagneticparticles can be held in the chamber by magnetic force and theuncaptured sample can be removed as waste, e.g., through a microfluidicchannel. Valves in the microfluidic device can then be configured toroute a volume, e.g., a non-microfluidic volume, of wash solution intothe chamber. The analyte and particles can be washed, immobilized andthe waste can be removed. Then, the particles with captured and purifiedanalyte can be re-suspended in a volume and moved from the chamberthrough a microfluidic channel into a thermal cycling chamber, which canbe a non-microfluidic reaction chamber or a microfluidic reactionchamber. There, the particles can be immobilized and liquid removed.This can concentrate the particles to about the particle volume (e.g.,in the nanoliter range). Reagents for performing nucleic acidamplification in an appropriate volume can be routed through amicrofluidic channel into the thermal cycling chamber. Under certainconditions, the reaction mixture will elute the nucleic acids from theparticles. Accordingly, this invention contemplates contacting reagentsfor performing a chemical reaction, such as PCR, cycle sequencing,real-time PCR, rolling circle amplification, restriction digestion, RNAfragmentation, protein digestion, etc., with the analyte attached to aparticle, without first eluting the analyte from the particle and, e.g.,determining concentration and selecting an amount of analyte for mixingwith reagent. This is possible, in part, by properly calibrating thecapture agent used to capture analyte, so that an appropriate amount ofanalyte is already present. After enzymatic reaction or thermal cycling,the product can be moved into a microfluidic channel for clean up, e.g.,in a microfluidic chamber or into a non-microfluidic chamber. This cancomprise diluting the product to decrease salt concentration. It alsocan comprise binding the analytes to beads, immobilizing them, andwashing them. It can also comprise chromatography, solid phaseextraction, or other cleanup methods at microfluidic scale ornon-microfluidic scale. At this point, the microfluidic components canroute the product to an appropriate location for analysis, e.g., acapillary tube for capillary electrophoresis or the product can beoutput into a non-microfluidic volume for analysis by an unintegratedsystem such as a commercial capillary array electrophoresis instrument,mass spectrometer, or other analytical instrument. The microfluidicdevice also can contain a waste confinement area.

In certain embodiments, the microfluidic devices of this invention aremonolithic devices. In monolithic devices, a plurality of circuits areprovides on a single substrate. In the case of devices comprisingdiaphragm valves, a monolithic device comprises a single elastic layerfunctioning as a diaphragm for a plurality of valves. In certainembodiments, one actuation channel can operate a plurality of valves ona monolithic device. This allows parallel activation of many fluidiccircuits. Monolithic devices can have dense arrays of microfluidiccircuits. These circuits function with high reliability, in part becausethe channels in each circuit are fabricated simultaneously on a singlesubstrate, rather than being made independently and assembled together.

The fluidic circuits and of these devices can be densely packed. Acircuit comprises an open or closed fluid conduit. In certainembodiments, the device can comprise at least 1 fluidic circuit per 1000mm², at least 2 fluidic circuits per 1000 mm², at least 5 fluidiccircuits per 1000 mm², at least 10 fluidic circuits per 1000 mm², atleast 20 fluidic circuits per 1000 mm², at least 50 fluidic circuits per1000 mm² Alternatively, the device can comprise at least 1 mm of channellength per 10 mm² area, at least 5 mm channel length per 10 mm², atleast 10 mm of channel length per 10 mm² or at least 20 mm channellength per 10 mm² Alternatively, the device can comprise valves (eitherseated or unseated) at a density of at least 1 valve per cm², at least 4valves per cm², or at least 10 valves per cm². Alternatively, the devicecan comprise features, such as channels, that are no more than 5 mmapart edge-to-edge, no more than 2 mm apart, no more than 1 mm apart, nomore than 500 microns apart or no more than 250 microns apart.

Universal Sample Preparation System

This invention provides a universal sample preparation system. Thesystem is configured to accept a biological sample comprising in analytein an un-purified form, purify the analyte, perform at least onebiochemical reaction on the analyte, and analyze the product of thebiochemical reaction.

The universal sample preparation system can be configured to fit insidea volume of no more than 6 ft³, no more than 7 ft³, no more than 8 ft³,no more than 97 ft³, or no more than 10 ft³. In one specific embodiment,the universal sample preparation system is configured to fit inside avolume of about 8 ft³. In another specific embodiment, the universalsample preparation system is configured to fit inside a volume of about10 ft³.

The universal sample preparation system can comprise the followingmodules: a sample preparation module, a reaction module, a post-reactionmodule, an analysis module, and/or a computer module. A samplepreparation module can be configured to capture in analyte, e.g., DNA,from a sample that has a non-microfluidic volume, and move the capturedanalyte into a first microfluidic channel. This can be accomplishedusing, for example, magnetically-responsive capture particles toconcentrate the analyte. The reaction module is fluidly connected to thefirst microfluidic channel and is configured to perform at least onebiochemical reaction on the analyte in a reaction chamber to produce areaction product. Typically, the analyte in the reaction chamber isconcentrated with respect to its concentration in the sample preparationmodule. A post-reaction module is in fluid communication with thereaction module and is configured to process the reaction product foranalysis. The post-reaction module can be configured to move thereaction product through a second microfluidic channel into anon-microfluidic chamber in the post-reaction module. The analysismodule is fluidly connected to the reaction module or the post-reactionmodule and is configured to perform an analysis, such as capillaryelectrophoresis, on the reaction product. The analysis module generatesdata about the reaction product. A computer module comprises computerexecutable code for processing and/or analyzing the data, includingformatting the data into a universal format. The system can be furtherconfigured to access the Internet, transmit data to an off-site serverand receive information from the server.

An exemplary device is shown in FIG. 6. FIG. 6 shows a cartridge withintegrated microchip (1), temperature modulating device (400), anddownstream analysis device (500). In certain embodiments the devicecomprises a fluid preparation module comprising a cartridge mated orotherwise fluidically connected to a microchip; an off-chip thermalmodulation module connected to the fluid preparation module through afluid transporter with a fluidic channel, such as a tube, through thecartridge, and configured to modulate the temperature in the fluidtransporter, wherein the fluid transporter is further fluidicallyconnected to a second microchip with valves and fluidic channels thatcan selectively route fluid to one or more subsequent devices. Thisdevice can be used for thermal cycling or isothermal reactions.

In one application, the system is configured to prepare, from abiological sample, a CODIS-compatible file for searching a CODISdatabase. CODIS is a forensic system that presently uses thirteen STR(short tandem repeat) loci as genetic identity markers see, e.g.,www.fbi.gov/hq/lab/html/codis1.htm. Information from fewer or more than13 markers can be used to provide evidence of identity. The samplepreparation module extracts DNA from forensic material, captures apredetermined amount of DNA on capture particles e.g. in magneticallyresponsive particles, and delivers the DNA to a non-microfluidicreaction chamber of a reaction module, where the particles areimmobilized, e.g., with a magnetic force. There, reagents for PCR aredelivered to the reaction chamber and the STR markers used in CODIS areamplified using PCR. The amplification can be performed usingcommercially available reagents, such as those available from Promega(e.g., the PowerPlex product series). This product uses four differentlycolored fluorescent markers. The PCR products are routed to thepost-reaction module and diluted to a proper concentration for capillaryelectrophoresis. The new product is directed to the analysis module,where capillary electrophoresis is performed. Analytes in the productare detected by fluorescence detection. The data generated is thensubjected to an algorithm which generates traces for each fluorescentspecies. These traces can then be analyzed by computer to identify themarkers at the STR loci used in CODIS. Such software is commerciallyavailable from, e.g., Life Technologies, such as GeneMapper™ ID-Xsoftware or NIST software tools(www.cstl.nist.gov/biotech/strbase/software.htm). Software can convertthis data into a file format compatible with CODIS. See, e.g.,www.ncjrs.gov/pdffiles1/nij/sl413apf.pdf. The CODIS compatible file canbe used to query a forensic database such as the CODIS database. Thesystem can be configured to access the Internet and deliver the file tothe appropriate database for analysis and to receive a report from thedatabase indicating the probable matches between the initial sample andan individual.

I. Sample Preparation Module

The sample preparation module is configured to capture an analyte from anon-microfluidic volume onto a solid substrate and route the capturedanalyte through a microfluidic channel into a reaction chamber of thereaction module. The analyte can be captured from a sample in which theanalyte is in non-isolated form. An analyte is non-isolated if it ispresent with other cellular or biomolecular components with which it isnaturally associated. These can include for example, cytoplasmiccomponents or viral components. The sample preparation module can beconfigured to capture and analyte from a raw sample. A raw sample is onein which the analyte is contained within a cell or a virus. For example,blood, a cheek swab and semen are raw samples containing the analyteDNA. In this case, the sample preparation module can comprise materialsto lyse cells and extract DNA from the lysate.

In one aspect a sample preparation device, as shown in FIG. 16, device1000 in FIG. 21 and FIG. 22, and FIG. 54, comprises a cartridgeintegrated with a microfluidic microchip that controls movement of thefluid in the cartridge through microvalves and the components to operatethe cartridge. The cartridge and/or the compartments therein can be ofsufficient size to process one or more milliliter of an input sample inan automated device. The cartridge can process a sample to output acomponent that can be moved using pressure-driven flow or vacuummodulated by microvalves. The cartridge can provide an interface with adelivery device comprising macroscale samples, such as blood, aerosolliquids, swabs, bodily fluids, swipes, and other liquid, solid, and gassamples. The cartridge can process macroscale sample volumes usingmicroscale sample preparation and analysis. The cartridge can allow forprocessing of macroscale or large volume samples using microfluidicdevices and components have reduced void volumes that allow for reducedloss of materials.

A. Cartridges

A cartridge, also referred to as a fluidic manifold herein, can be usedfor a number of purposes. In general, a cartridge can have ports thatare sized to interface with large scale devices as well as microfluidicdevices. Cartridges or fluidic manifolds have been described in U.S.Patent Publication US20090253181. The cartridge can be used to receivematerials, such as samples, reagents, or solid particles, from a sourceand deliver them to the microfluidic microchip. The materials can betransferred between the cartridge and the microfluidic microchip throughmated openings of the cartridge and the microfluidic microchip. Forexample, a pipette can be used to transfer materials to the cartridge,which in turn, can then deliver the materials to the microfluidicdevice. In another embodiment, tubing can transfer the materials to thecartridge. In another embodiment, a syringe can transfer material to thecartridge. In addition, a cartridge can have reservoirs with volumescapable of holding nanoliters, microliters, milliliters, or liters offluid. The reservoirs can be used as holding chambers, reaction chambers(e.g., that comprise reagents for carrying out a reaction), chambers forproviding heating or cooling (e.g., that contain thermal controlelements or that are thermally connected to thermal control devices), orseparation chambers (e.g. paramagnetic bead separations, affinitycapture matrices, and chromatography). Any type of chamber can be usedin the devices described herein, e.g., those described in U.S. PatentPublication Number 2007/0248958, which is hereby incorporated byreference. A reservoir can be used to provide heating or cooling byhaving inlets and outlets for the movement of temperature controlledfluids in and out of the cartridge, which then can provide temperaturecontrol to the microfluidic microchip. Alternatively, a reservoir canhouse Peltier elements, or any other heating or cooling elements knownto those skilled in the art, that provide a heat sink or heat source. Acartridge reservoir or chamber can have a volume of at least about 0.1,0.5, 1, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 750, 1000, 2000,3000, 4000, 5000 or more μL. The relative volume of a chamber orreservoir can be about 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000,5000, 10000 or more greater than a channel or valve within themicrofluidic microchip. The size of the chambers and reservoirs of thecartridge, which can be mated to the microfluidic microchip, can bechosen such that a large volume of sample, such as a sample greater thanabout 1, 5, 10, 50, 100, 500, 1000, 5000, 10000, 50000 or more μL, canbe processed, wherein the flow of fluids for processing the sample iscontrolled by valves in the microfluidic microchip. This can allow for areduced amount of sample and reagent loss due to the reduced voidvolumes in the microfluidic microchip compared to other flow controldevices, such as pipettes and large scale valves. The void volume withina microfluidic microchip can be less than 1000, 500, 100, 50, 10, 5, 1,0.5, 0.1, or 0.05 μL. This can allow for the amount of sample or reagentloss during processing of a sample to be less than 20, 15, 10, 7, 5, 3,2, 1, 0.5, 0.05 percent.

A cartridge can be constructed of any material known to those skilled inthe art. For example, the cartridge can be constructed of a plastic,glass, or metal. A plastic material may include any plastic known tothose skilled in the art, such as polypropylene, polystyrene,polyethylene, polyethylene terephthalate, polyester, polyamide,poly(vinylchloride), polycarbonate, polyurethane, polyvinyldienechloride, cyclic olefin copolymer, or any combination thereof. Thecartridge can be formed using any technique known to those skilled inthe art, such as soft-lithography, hard-lithography, milling, embossing,ablating, drilling, etching, injection molding, or any combinationthereof.

As exemplified in FIG. 3 and FIG. 4, a cartridge (1) can comprise arectilinear configuration with flat sides. In another embodiment, acartridge comprises a surface that is curved, rounded, indented orcomprises a protrusion. In one embodiment a cartridge has at least onesubstantially flat surface which is adjacent to a microfluidicmicrochip. The cartridge is adapted to be fluidically connected withports in the microchip. For example, openings in the surface of thecartridge can be aligned with ports in the microchip. When the cartridgeand microchip are mated to one another, the openings align to create thefluidic connections allowing liquids to pass from the cartridge into theports of the microchip, which are connected to channels typically havingvalves that form fluidic circuits.

In one embodiment a cartridge contains one or more features, includingbut not limited to a chamber, a port, a channel or a magnet. In oneembodiment, microvalves, such as pneumatically actuated valves arecombined with the microfluidic cartridge. In some embodiments themicrovalves are active mechanical microvalves (such as magnetic,electrical, or piezoelectric thermal microvalves), non-mechanicalmicrovalves (such as bistable electromechanical phase change orrheological microvalves), external microvalves (such as modular orpneumatic), passive mechanical (such as check microvalves or passivenon-mechanical (such as capillary microvalves) (Oh et al., A review ofmicrovalves, J. Micromech Microeng. 16 (2006) R13-R39)).

In another embodiment, pneumatically actuated valves, such as MOVevalves, modulate the flow of air pressure, vacuum, or fluids in amicrofluidic microchip 2 or multiple microfluidic microchips. MOVevalves can be microscale on-chip valves, microfluidic on-chip valves ormicro-robotic on-chip valves. In one embodiment the flow of airpressure, vacuum, or fluids is regulated by one or more variablepressure pumps, such as solenoid valves or solenoid pumps. In oneembodiment, a microfluidic microchip is a structure that containsmicrochannels and/or microtrenches, where a microchannel is a closedstructure and a microtrench is an open structure. In one embodiment amicrofluidic microchip is a planar structure. In a related embodiment amicrofluidic device comprises a microfluidic microchip with microvalvesclustered on one side of a cartridge. In one embodiment (FIG. 3 and FIG.4) the cartridge (1) can comprise one or more ports (4, 5, 6, 7, 8, 9)to external fluids, air, or vacuum. Functions of the ports can be forwaste (4), reagent entry (5), vent (6), sample input (7), product output(8). The cartridge (1) can contain one or more sample input or reactionchambers, (7) and (3).

A single chamber within the cartridge, such as a reaction chamber, canhave one or more, or at least one, two, or three fluidic connections toa microchip. For example, in FIG. 3 and FIG. 4, the reaction chamber (3)can have a fluidic connection to the microchip through connection 120,which is at the base of the chamber, and another fluidic connection tothe microchip through port (9), which is connected to chamber (3)through a passageway located at the top of the chamber. The top ofchamber (3), port (9), and the passageway between chamber (3) and port(9) can be closed from the exterior environment such that fluids inchamber (3) necessarily are pumped into port (9) when chamber (3) isfull and vice versa. Such a chamber or combination or chamber and portcan be referred to as a closed chamber. The positioning of the fluidicconnections need not necessarily be at the base and top of the chamber,however, fluidic connections at the base and top positions of thechamber allow for reduced trapping of gas in the chamber. Alternatively,reaction chamber (3) can be viewed as a combination of two chambers thatare fluidically connected to each other at a top position, which can bewithin the cartridge, and, where each chamber also has an opening at abase location. The openings at the base locations, also called chamberapertures, can be fluidically connected to port apertures on themicrochip. The two fluidic connections can allow for fluids to bedirected into and out of the chamber through the microfluidic microchip.

In another embodiment a device comprises a cartridge comprising at leastone pneumatically actuated valve, such as a MOVe valve, located on oneor more surfaces or structures in a non-linear manner. A cartridge cancomprise one or more pneumatically actuated valves that are locatedwithin the cartridge, in a location other than the base of thecartridge.

Functional elements of a cartridge can include ports, channels,chambers, filters, magnets, or vents, chambers can be collectivelyreferred to as functional elements. In one embodiment, FIG. 4, thefunctional elements connect to the microfluidic microchip containingmicrovalves at junctions 100, 120, 140, 160, and 230. The functionalelements can connect with tubing or capillaries inserted into the ports,by a flush connection, or by fittings. In one embodiment a flushconnection can comprise a port of a cartridge aligned directly with anaperture of a microfluidic microchip. In one embodiment the cartridgeand microfluidic microchip form an integrated module. In anotherembodiment the cartridge and microfluidic microchip are two separatepieces which are attached together, prior to use.

A cartridge can comprise at least one chamber, a sample input port, areagent port, an exit port, a waste port and a magnet. The magnet can belocated adjacent to the chamber, so that the magnet force generated bythe magnet can attract paramagnetic particles in said chamber to a wallof the chamber. In one embodiment the paramagnetic particles are beadsor cells rendered magnetically responsive (e.g., cells comprisinghemoglobin that are treated with sodium nitrate). The magnet can be anelectric magnet or a permanent magnet, such as a rare earth metalmagnet.

In one embodiment, as exemplified in FIG. 4, connections or ports (4, 5,6, 7, 8, and 9) lead to channels in the cartridge (14, 15, 16, 17, 18,and 19) respectively. Ports (4, 5, 6, 7, and 8) show indents to reliablyattach a connector or tubing to the indent, such as the indent shown forconnection (7) (see the difference in diameter of connection (7) withchannel (17)). In one embodiment, the ports or ports can interface witha variety of connector or tubes, such as the capillaries as described inU.S. Pat. No. 6,190,616, U.S. Pat. No. 6,423,536, U.S. application Ser.No. 09/770,412, Jan. 25, 2001, U.S. Application No. 60/402,959 or one ormore microchips with modular microfluidic ports as described in U.S.Pat. No. 6,870,185 and U.S. application Ser. No. 11/229,065; all ofwhich are herein incorporated by reference in their entirety. In oneembodiment, the modular microfluidic ports enable microchips orcapillaries to be reversibly joined without dead volumes or leakage.

In another embodiment chamber (3) is connected to passageway (9) and tocone (13), leading to junction (120). Chamber (3) can be used forreactions as may any of the channels. In FIG. 4 the cartridge channelslead directly to the apertures of ports on the microchip (2). Thechannels of the cartridge can interconnect with each other as needed. Insome embodiments, at least one channel in a cartridge does notphysically connect to a microfluidic microchip. In another embodiment atleast one channel in a cartridge is fluidically connected to at leastone microchannel in a microfluidic microchip. The connection may or maynot utilize an aperture on the microfluidic microchip. An aperture canbe an opening or a fitting designed to mate between the microchip andthe cartridge. In some embodiments of the invention, the fittingcomprises a seal such as a gasket or an o-ring.

B. Microfluidic Devices

In one embodiment a cartridge and a microfluidic microchip areintegrated together to form a single modular device. The cartridge and amicrofluidic microchip can be attached by a fluid or by a solid adhesiveor mechanically. In one embodiment the adhesive is a polyacrylate,adhesive tape, double-sided tape, or any other adhesive known to oneskilled in the art. A cartridge can comprise a feature (12), asexemplified in FIG. 4, that is capable of wicking a fluid-based adhesiveinto the junction between a microfluidic microchip and a cartridge. Anexemplary In another embodiment a cartridge is attached to amicrofluidic microchip with a non-fluidic adhesive layer. Alternatively,the cartridge and microchip can be held together by clips, clamps, oranother holding device. The cartridge and microchip can be aligned priorto integration by visual cues, with or without a microscope, or byphysical guiding features. Visual cues can include lines or featuresthat are drawn, etched, or otherwise present on the cartridge, themicrochip, or both. Physical guiding features include indentations,protrusions, and edges that can be ‘keyed’ to aid or insure properassembly.

In some instances, the microfluidic microchip has diaphragm valves forthe control of fluid flow. Microfluidic devices with diaphragm valvesthat control fluid flow have been described in U.S. Pat. No. 7,445,926,U.S. Patent Publication Nos. 2006/0073484, 2006/0073484, 2007/0248958,and 2008/0014576, and PCT Publication No. WO 2008/115626. The valves canbe controlled by applying positive or negative pressure to a pneumaticslayer of the microchip through a pneumatic manifold.

In one embodiment, the microchip is a “MOVe” microchip. Such microchipscomprise three functional layers—a fluidics layer that comprisesmicrofluidic channels; a pneumatics layer that comprises pneumaticschannels and an actuation layer sandwiched between the two other layers.In certain embodiments, the fluidics layer is comprised of two layers.One layer can comprise grooves that provide the microfluidics channels,and vias, or holes that pass from the outside surface to a fluidicschannel. A second layer can comprise vias that pass from a surface thatis in contact with the actuation layer to the surface in contact withthe pneumatic channels on the other layer. When contacted together,these two layers from a single fluidics layer that comprises internalchannels and vias that open out to connect a channel with the fluidicsmanifold or in to connect a channel with the activation layer, to form avalve, chamber or other functional item. The actuation layer typicallyis formed of a deformable substance, e.g., an elastomeric substance,that can deform when vacuum or pressure is exerted on it. At pointswhere the fluidic channels or pneumatic channels open onto or areotherwise in contact with the actuation layer, functional devices suchas valves, e.g. diaphragm valves, can be formed. Such a valve isdepicted in cross section in FIG. 1. Both the fluidics layer and thepneumatics layer can comprise ports that connect channels to the outsidesurface as ports. Such ports can be adapted to engage fluidicsmanifolds, e.g., cartridges, or pneumatics manifolds.

As shown in FIG. 40, the microfluidic microchip (103) can be interfacedwith the cartridge (101). The microfluidic microchip can have a chamber(105) with an opening that is mated to an opening (117) of the cartridge(101). The chamber can be used for a variety of purposes. For example,the chamber can be used as a reaction chamber, a mixing chamber, or acapture chamber. The chamber can be used to capture magnetic particlessuch as magnetic beads, paramagnetic beads, solid phase extractionmaterial, monoliths, or chromatography matrices.

A magnetic component (109) can be positioned such that magneticparticles in the cartridge reservoir (107) and/or the microfluidicchamber (105) are captured against a surface of the microfluidic chamber(105). The magnetic component can generate a magnetic and/orelectromagnetic field using a permanent magnet and/or an electromagnet.If a permanent magnet is used, the magnet can be actuated in one or moredirections to bring the magnet into proximity of the microfluidicmicrochip to apply a magnetic field to the microfluidic chamber. In someembodiments of the invention, the magnet is actuated in the direction(111) indicated in FIG. 40.

Alternatively, any of a variety of devices can be interfaced with themicrofluidic microchip. For example detectors, separation devices (e.g.gas chromatographs, liquid chromatographs, capillary electrophoresis,mass spectrometers, etc), light sources, or temperature control devicescan be positioned next to the microfluidic microchip or used inconjunction with the microfluidic microchip. These devices can allow fordetection of analytes by detecting resistance, capacitance, lightabsorbance or emission, fluorescence, or temperature or other chemicalor physical measurements. Alternatively, these devices can allow forlight to be introduced to a region or area of the microfluidicmicrochip.

A microfluidic device can be designed with multiple chambers that areconfigured for capture of magnetic particles. The multiple chambers andmagnetic component can be arranged such that a magnetic field can beapplied simultaneously to all chambers, or be applied to each or somechambers independent of other chambers. The arrangement of chambers andmagnetic components can facilitate faster or more efficient recovery ofmagnetic particles. In particular, the arrangement can facilitaterecovery of magnetic particles in multiple chambers.

As shown in FIG. 41, the microfluidic microchip (103) can be formed of afluidics layer (203), an elastomeric layer (205), and a pneumatic layer(207). The fluidics layer can contain features such as a chamber (105),as well as channels, valves, and ports. The channels can be microfluidicchannels used for the transfer of fluids between chambers and/or ports.The valves can be any type of valve used in microfluidic devices. Inpreferred embodiments of the invention, a valve includes a microscaleon-chip valve (MOVe), also referred to as a microfluidic diaphragm valveherein. A series of three MOVes can form a MOVe pump. The MOVes and MOVepumps can be actuated using pneumatics. Pneumatic sources can beinternal or external to the microfluidic microchip.

An example of a MOVe valve is shown in FIG. 1. A clamshell view of aMOVe valve is shown in FIG. 1A. A cross-sectional view of a closed MOVevalve is shown in FIG. 1B. FIG. 1C shows a top-down view of the MOVevalve. A channel (251) that originates from a fluidic layer caninterface with an elastomeric layer (259) by one or more vias (257). Thechannel can have one or more seats (255) to obstruct flow through thechannel when the elastomeric layer (259) is in contact with the seat(255). The elastomeric layer can either be normally in contact with theseat, or normally not in contact with the seat. Application of positiveor negative pressure through a pneumatic line (261) to increase ordecrease the pressure in a pneumatic chamber (253) relative to thefluidic channel (251) can deform the elastomeric layer, such that theelastomeric layer is pushed against the seat or pulled away from theseat.

In some embodiments of the invention, a MOVe does not have a valve seat,and fluid flow through the fluidic channel is not completely obstructedunder application of positive or negative pressure. This type of valveis useful as a fluid reservoir and as a pumping chamber and can bereferred to as a pumping valve. The vacuum that can be applied includeextremely high vacuum, medium vacuum, low vacuum, house vacuum, andpressures such as 5 psi, 10 psi, 15 psi, 25 psi, 30 psi, 40 psi, 45 psi,and 50 psi.

Three MOVe valves in series can form a pump through the use of a firstMOVe as an inlet valve, a second MOVe as a pumping valve, and a thirdMOVe as an outlet valve. Fluid can be moved through the series of MOVesby sequential opening and closing of the MOVes. For a fluid beingsupplied to an inlet valve, an exemplary sequence can include, startingfrom a state where all three MOVes are closed, (a) opening the inletvalve, (b) opening the pumping valve, (c) closing the inlet valve andopening the outlet valve, (d) closing the pumping valve, and (e) closingthe outlet valve. Since the inlet and outlet valve can have the samestructure, a MOVe pump can move fluids in either direction byreprogramming of the sequence of opening inlet or outlet valves.

The fluidic layer (203) can be constructed of one or more layers ofmaterial. As shown in FIG. 42, the fluidic layer (203) can beconstructed of two layers of material. Channels (301, 303, 305) can beformed at the interface between the two layers of material, and achamber (105) can be formed by complete removal of a portion of onelayer of material. The channels can have any shape, e.g., rounded and onone side (301), rectangular (303), or circular (305). The channel can beformed by recesses in only one layer (301, 303) or by recesses in bothlayers (305). The channels and chambers can be connected by fluidicchannels that traverse the channels and chambers shown. Multidimensionalmicrochips are also within the scope of the instant invention wherefluidic channels and connections are made between multiple fluidiclayers.

The thickness (307) of the second layer of material can be of anythickness. In some embodiments of the invention, the second layer has athickness that minimizes reduction of a magnetic field in the chamber(105) that is applied across the second layer from an external magneticcomponent or minimizes reductions in heat transfer.

As shown in FIG. 43, the fluidic layer (203) can be constructed of asingle layer of material. The single layer is then interfaced with anelastomeric layer (205), such that channels (305, 303) and chambers(305) are formed between the fluidic layer and the elastomeric layer.

The microfluidic microchip can be constructed from any material known tothose skilled in the art. In some embodiments of the invention, thefluidics and pneumatic layer are constructed from glass and theelastomeric layer is formed from PDMS. In alternative embodiments, theelastomer can be replaced by a thin membrane of deformable material suchas Teflon (PTFE), silicon, or other membrane. The features of thefluidics and pneumatic layer can be formed using any microfabricationtechnique known to those skilled in the art, such as patterning,etching, milling, molding, embossing, screen printing, laser ablation,substrate deposition, chemical vapor deposition, or any combinationthereof.

Microfluidic devices can be configured so that valves are less sticky.This can be accomplished by coating valve seats and other surfaces overwhich fluid flows that are likely to come into contact with the elasticlayer with low energy material, such a noble metal (e.g., gold) or aperfluorinated polymer (e.g., Teflon). Such devices are described inmore detail in U.S. patent application Ser. No. 12/789,186.

In one embodiment, microchannel circuits are formed on a microfluidicmicrochip 2, as shown in FIG. 5, linking sets of microvalves withmicrochannels. In one embodiment the microvalves are pneumaticallyactuated valves. In one embodiment the pneumatically actuated valves areMOVe microvalves. In one embodiment, the fluidic path between acartridge and a microfluidic microchip, such as between chambers, ports,channels, microchannels, and other functional elements can be controlledby opening or closing at least one microvalve. In one embodiment themicrovalve is controlled by a microprocessor control such as a computer.A computer can include an input/output controller, or any othercomponents known to one skilled in the art such as memory storage and aprocessor. In one embodiment, a microvalve is a MOVe valve that isactuated by a pneumatic source, such as through pneumatic ports 10, 20,30, 40, 50, 60, or 70. In one embodiment the pneumatic source iscontrolled by at least one solenoid. In one embodiment the solenoid isminiaturized and can be connected to vacuum or pressure sources. In oneembodiment the pneumatic source is connected to a pneumatic port using aforce such as clamping, springs, pneumatics, or a screw force,optionally with sealing provided by an o-ring.

In one embodiment FIG. 5 shows a view of the top of a microfluidicmicrochip (2), this side makes contact with the bottom of cartridge (1).A microvalve 110 controls the fluidic path between microchannels 101 and121. A microvalve 130 controls the fluidic path between microchannels131 and 141. Microvalve (150) controls the fluidic path betweenmicrochannels 151 and 152. Microvalve 180 controls the fluidic pathbetween microchannels 181 and 191. Microvalve 200 controls the fluidicpath between microchannels 201 and 212. Microvalve 220 controls thefluidic path between microchannels 221 and 231.

In one embodiment junctions can connect one or more microchannels. FIG.5 shows the schematic for a microchip that can be mated with thecartridge shown in FIG. 4. In FIG. 5, junction 100 connects to singlemicrochannel 101, junction 140 connects to single microchannel 141,junction 160 connects to single microchannel 161, and junction 230connects to single microchannel 231. Junction 190 connects to twomicrochannels 191 and 201. Junction 120 connects to three microchannels121, 131, and 151. In one embodiment more than three microchannels canbe connected to a single junction.

The microchannels can be fabricated by one or more techniques such asphotolithography, molding, embossing, casting, or milling. Themicrochannels can be manufactured in a material such as glass, plastic,polymer, ceramic, gel, metal, or another suitable solid.

In one embodiment the cartridge is used in a method of sample enrichmentcomprising: delivery of a sample to a chamber by a sample port anddelivery of paramagnetic particles to a chamber by a reagent port. Theparamagnetic particles (e.g. paramagnetic beads) bind to at least onecomponent in the sample (such as DNA, RNA, micro RNA, a protein, alipid, a polysaccharide or other ligand). The paramagnetic particles areattracted to a wall of a chamber by virtue of the magnetic force exertedby a magnet located outside the chamber. The paramagnetic particles arewashed with a wash solution delivered to the chamber comprising theparamagnetic particles by a reagent port, and the wash solution isremoved by a waste port. A reagent can be added to elute the componentof the sample from the paramagnetic particles and output the samplecomponent to another device for further processing or analysis. Apreferred embodiment is to output the component of the sample on theparamagnetic particles.

In one embodiment a device comprising a microfluidic microchip is usedin a method of diagnosis. In one embodiment the diagnosis comprises thedetection of an infectious agent in a sample. In one embodiment theinfectious agent is a bacteria, virus, fungi, mycoplasm or prion. Inanother embodiment a device comprising a microfluidic microchip is usedin a method of diagnosis of a hereditary disease. In one embodiment thehereditary disease is caused by one or more DNA mutations, suchmutations include but are not limited, triplet base expansions, basesubstitution mutations, deletion mutations, addition mutations, nonsensemutations, premature stop codons, chromosomal deletions, chromosomalduplications, aneuploidy, partial aneuploidy or monosomy. In anotherembodiment a device comprising a microfluidic microchip is used in amethod to diagnose cancer or a predisposition to cancer. In anotherembodiment a device comprising a microfluidic microchip is used in amethod to diagnose a hereditary disease such as autism, downs syndrome,trisomy, Tay-sachs, or other hereditary diseases. In some embodiments asample used for diagnosis in a device comprising a microfluidicmicrochip is a blood sample, a mucus sample, a lung lavage sample, aurine sample, a fecal sample, a skin sample, a hair sample, a semensample, a vaginal sample, or an amniotic sample.

In another embodiment a device comprising a microfluidic microchip isused to identify the presence of environmental contamination of anagent. In one embodiment the agent is a biological agent such asbacteria, virus, fungi, or mycoplasm in an environmental sample. Inanother embodiment the agent is a contaminant agent, such as apesticide, an herbicide, or a fertilizer. In one embodiment theenvironmental sample is a soil sample, a water sample, an air sample, ameat sample, a vegetable sample or a fruit sample. In anotherembodiment, the agent is a genetically modified organism.

In another embodiment a device comprising a microfluidic microchip isused for genotyping, identification of an individual mammal (such as ahuman), forensics, gene expression, gene modification, microRNAanalysis, or ribotyping.

In another embodiment a device comprising a microfluidic microchip isused in a method comprising molecular biological analysis, including butnot limited to polymerase chain reaction (PCR) amplification of nucleicacids in a sample (such as Allele-specific PCR, Assembly PCR, AsymmetricPCR, Colony PCR, Helicase-dependent amplification, Hot-start PCR,Intersequence-specific (ISSR) PCR, Inverse PCR, Ligation-mediated PCR,Methylation-specific PCR Multiplex Ligation-dependent ProbeAmplification, Multiplex-PCR, Nested PCR, Overlap-extension PCR,Quantitative PCR Reverse Transcription PCR-PCR, Thermal asymmetricinterlaced-PCR, Touchdown PCR, or PAN-AC PCR), isothermal nucleic acidamplifications, (such as Loop-mediated Isothermal Amplification (LAMP);nick displacement amplification; Helicase Dependant Amplificationplatform (HDA); and the primase-based Whole Genome Amplificationplatform (pWGA); single primer isothermal amplification (SPIA) andRibo-SPIA for RNA; strand displacement amplification (SDA); EXPAR [VanNess J, Van Ness L K, Galas D J. (2003) Isothermal reactions for theamplification of oligonucleotides. Proc Natl Acad Sci USA. 100:4504-9.];rolling circle amplification (RCA); transcription-based amplificationsystem (TAS) and its derivatives include self-sustaining sequencereplication (3SR), isothermal nucleic acid sequence-based amplification(NASBA), and transcription-mediated amplification (TMA); ligase chainreaction (LCR)), sequencing reactions of DNA or RNA (such asMaxam-Gilbert sequencing, Sanger chain-termination method,Dye-terminator sequencing Emulsion PCR sequencing, massively parallelsequencing, polony sequencing, sequencing by ligation, sequencing bysynthesis, or sequencing by hybridization), restriction fragment lengthpolymorphism (RFLP) analysis, single nucleotide polymorphism (SNP)analysis, short tandem repeat (STR) analysis, microsatellite analysis,DNA fingerprint analysis, DNA footprint analysis, or DNA methylationanalysis.

In one embodiment a cartridge employs beads coupled to a binding moiety,including but not limited to a binding receptor, transferrin, anantibody or a fragment thereof (such as an Fc fragment or an Fabfragment), a lectin, or a DNA or RNA sequence. In another embodiment acartridge comprises a reagent such as an anti- coagulant, a fixative, astabilization reagent, a preservative or precipitation reagent.

C. Pneumatic Manifold

A pneumatic manifold can be integrated with any microchip and/orcartridge described herein to facilitate distribution of air pressure orvacuum. The air pressure or vacuum can be used to actuate valves on themicrochip. Alternatively, air pressure or vacuum can be supplied to acartridge such that air pressure or vacuum is provided to microchannelswithin the fluidics layer of a microchip which can be used to movefluids or gases within the fluidics layer. A pneumatic manifold providesthe air pressure or vacuum to operate microvalves on microchip (2) oncartridge (1) of FIG. 3 or operate microvalves in other devices.

A pneumatic manifold can be used to mate the pneumatic lines of amicrofluidic microchip to external pressure sources. The pneumaticmanifold can have ports that align with ports on the pneumatics layer ofthe microfluidic microchip and ports that can be connected to tubingthat connect to the external pressure sources. The ports can beconnected by one or more channels that allow for fluid communication ofa liquid or gas, or other material between the ports.

The pneumatic manifold can be interfaced with the microfluidic microchipon any surface of the microchip. The pneumatic manifold can be on thesame or different side of the microfluidic microchip as the cartridge.As shown in FIG. 40, a pneumatic manifold (113) can be placed on asurface of the microfluidic microchip opposite to the cartridge. Aswell, the pneumatic manifold can be designed such that it only occupiesa portion of the surface of microfluidic microchip. The positioning,design, and/or shape of the pneumatic manifold can allow access of othercomponents to the microfluidic microchip. The pneumatic manifold canhave a cut-out or annular space that allows other components to bepositioned adjacent or proximal to the microfluidic microchip. This canallow, for example, a magnetic component (109) to be placed in proximityof a chamber within the microfluidic microchip.

A pneumatic manifold can be constructed of any material known to thoseskilled in the art. For example, the cartridge can be constructed of aplastic, glass, or metal. A plastic material includes any plastic knownto those skilled in the art, such as polypropylene, polystyrene,polyethylene, polyethylene terephthalate, polyester, polyamide,poly(vinylchloride), polycarbonate, polyurethane, polyvinyldienechloride, cyclic olefin copolymer, or any combination thereof. Thepneumatic manifold can be formed using any technique known to thoseskilled in the art, such as soft-lithography, conventional lithography,milling, molding, embossing, drilling, etching, or any combinationthereof.

The apparatus shown in FIG. 21 and FIG. 22 can incorporate a pneumaticmanifold. The apparatus can be used for sample preparation, as describedherein, and can incorporate a cartridge. Cartridge (1), labeled ‘cube’,is attached to manifold (370) with solenoids (1819). The assembly of thecartridge and manifold is mounted on a base plate of the apparatus. Thepneumatic manifold can be controlled by an IO controller (1803).

A gas supply, such as a reservoir that can be maintained at a desiredpressure or vacuum, can supply gas to the manifold. The gas supply canbe connected to an outside pressure or vacuum source. The gas supplyfeeding the gas supply manifold can have a pressure gauge to monitor theinlet pressure. The gas supply can supply gas to multiple components ofthe system through a gas supply manifold (1821). The gas supply manifoldcan supply gas to the pneumatic manifold (370) and to individual reagentcontainers, (1809) and (1807). The line supplying the distribution valve(390) with gas can be regulated by a regulator (1815).

Reagents and/or sample can also be supplied to the cartridge through thereagent distribution valve (390) that is connected to containers (1809)in a reagent storage region (380) and a bead solution container (1807)that is mounted on a bead mixer (1805). Adapter (1817) can be mountedand/or aligned with the cartridge such that a delivery device, such as asyringe, can deliver a material to the cartridge. The adapter (1817) canbe thermally regulated by a heater control (1801). The adapter can havea thermal conductor, such as brass, to distribute heat generated byheater coil or a Peltier device. The adapter can maintain temperaturebetween about 20 to 100, 20 to 75, or 50 to 60 degrees Celsius.

A magnet assembly (1811) can be positioned adjacent to the cartridge. Amagnet (300) of the magnet assembly can be positioned adjacent to thecartridge (1) and moved by an actuator, such that the magnet can exert amagnetic field within the cartridge, or a microchip integrated, mated,or interfaced with the cartridge. The magnetic field can be used tocapture paramagnetic or magnetic particles, such as beads, within thecartridge or microchip and separate material bound to the particles fromwaste materials. Waste from the cartridge and/or microchip can bedelivered to a waste container (1813).

The apparatus shown in FIG. 21 and FIG. 22 can use seven solenoid valvesto operate the cartridge (1). The size and complexity of the apparatuscan be further reduced with MOVe microvalves. FIG. 23 and FIG. 24 show areagent distribution device that contains microfluidic microchip 600,which is approximately two inches wide. Solenoid banks 680 and 684provide connection to full scale external vacuum and pressure throughconnectors 681, 682, 685, and 686. The solenoids are controlled throughelectrical junctions 689 and 687. The microfluidic microchip 600, whichhas MOVe valves, is held in contact with the manifold 700 by attachment711 using clamp 710. Other methods known to one skilled in the art canbe used to connect the microchip to the pneumatics manifold 700.

D. Parallel Processing of Samples

In some embodiments of the invention, one or more cartridges can beoperated simultaneously to allow for parallel processing of samples.FIG. 16 illustrates parallel or ganged operation of multiple cartridgeswith microvalves on a single pneumatic manifold in swab extractionassembly (800). The manifold (370) distributes regulated vacuum andpressure to operate four cartridges (1), indicated in the figure, usingsolenoids (680). Solenoids (680) control pressure to the pneumatic layerof a microchip integrated with each cartridge through the pneumaticmanifold (370, 380, 390). The pneumatic manifold is formed by a topplate (370), a gasket (380) and a bottom plate (390). The top plate canhave channels etched into it. The channels can be sealed by the gasket,which is sandwiched against the top plate by the bottom plate (390).Actuator 310 moves rod 810 to move magnets (320) close to or away fromthe cartridges (1). Clamps 805 hold cartridges (1) in place.

In other embodiments of the invention, a single cartridge integratedwith a microchip can process multiple samples at one time using parallelchannels. For example, the device can be configured to have 4, 8, oreven 12 channels. FIG. 14 and FIG. 15 shows an assembled capture andreaction microchip with capillary feed and magnets. This microchip cancapture bead solutions and perform, for example, four, STR-PCR reactionssimultaneously. FIG. 14 shows a microchip (1201) with a cartridge (1203)adhered to the microchip and tubes (1205, 1207, 1209, 1211, 1213, 1215,1217, and 1219) leading into and out of the microchip. A total of eighttubes are shown and two tubes are used per parallel reaction. Forexample, one unit of the parallel processing device is served by tubes1205 and 1213.

In an exemplary embodiment, a four-channel sample preparation devicecombines a four-channel parallel reagent delivery device (FIG. 53) thatmeters and delivers reagents simultaneously to all four channels of asingle integrated cartridge (FIG. 54) enabling four samples to beprocessed simultaneously and rapidly.

The four-channel parallel reagent delivery device combines a microchip(see FIG. 53) with a fluidics manifold mounted on a pneumatics controlmanifold. FIG. 53 shows a sample extraction module comprises a cartridgemated to a microfluidic chip. The cartridge comprises apertures forsyringes (6701) and compartments for bead capture of analyte (6702).These compartments are fluidically connected through channels (6703) ina microfluidic chip mated to the undersurface of the cartridge. Areagent distribution cartridge can comprise compartments configuredsimilar to those in FIG. 3. The two can be fluidically connected, e.g.,by tubes, and reagents can be pumped from reagent reservoirs into thechambers on the reagent cartridge and then into the sample extractionmodule. 4, 8, 12 channel and devices with more channels arecontemplated. Reagents are metered, using one of the two different sizereagent loops, which can be similar to the sample loops describedherein, for each channel, and delivered in parallel to all four channelsof the sample preparation device. Delivering reagents simultaneously toall four channels of the sample preparation device using the parallelreagent delivery device can takes <4 minutes, representing a processtime saving of >11 minutes as compared to the first generation serialreagent delivery device that took ˜15 minutes per four samplesprocessed.

Bonded pneumatics manifolds can be used to control both the reagentdelivery and sample preparation devices by fabricating the manifoldsusing an adhesive bonding approach; however, these may be prone todelamination over time due to the pneumatic pressures used in thesubsystem, and the size and complexity of the manifold. Thermally bondedmanifolds can mitigate delamination issues, but may only be a viableapproach for relatively small and low complexity manifold designs suchas the reagent delivery device. A monolithic manifold made from a singlepiece of polycarbonate with tubing connecting pneumatic ports to thesolenoid control valves can operate the four-channel sample preparationcartridge and has proved to be a viable alternative to bonded pneumaticmanifolds. This pneumatic manifold design concept is also being utilizedfor control of the Chip A microchip on the Post-amplification STR (ShortTandem Repeat) clean-up subsystem.

Assembly processes for the microchip and fluidic manifold of thefour-channel sample preparation cartridge have also been improved.Historically, silicon epoxy can be used to attach the cartridge to itsassociated MOVe microchip by wicking the adhesive between the microchipand the cube. An inherent lack of control of the movement of the epoxycan allow it to occasionally wick into the ports on either the microchipor the cube creating a blockage in the fluidic pathway rendering thedevice unusable. This process has been improved by using a double-sidedadhesive tape (Adhesives Research ARcare90106) to assemble the fluidiccubes and microchips; this is now the preferred assembly method used forthe four-channel reagent delivery cartridge, the sample preparationdevice, and the post amplification device in the Post-amplification STRclean-up subsystem described below.

The integrated four-channel sample preparation cartridge with the 069microchip (see FIG. 55) was tested. The 069 microchip design is usedwith the sample extraction cartridge. Other chips, for example the 070chip is used with the post-amplification cartridge.

Microchip blockages due to the inadvertent introduction of fibers intothe systems and devices described herein can be problematic inmicrofluidics. To minimize blockages, all reagents with the exception ofparamagnetic bead solutions, can be filtered prior to loading andin-line filters used to minimize microchip blockages.

E. Separation and Cleanup

A variety of separations can be performed using the devices describedherein. These separations include chromatographic, affinity,electrostatic, hydrophobic, ion-exchange, magnetic, drag-based, anddensity-based separations. In some embodiments of the invention,affinity or ion-exchange interactions are utilized to bind materials tosolid-phase materials, such as beads. The beads can be separated fromfluid solutions using any method known to those skilled in the art.

Magnetic separation can be used to capture and concentrate materials ina single step using a mechanistically simplified format that employsparamagnetic beads and a magnetic field. The beads can be used tocapture, concentrate, and then purify specific target antigens,proteins, carbohydrates, toxins, nucleic acids, cells, viruses, andspores. The beads can have a specific affinity reagent, typically anantibody, aptamer, or DNA that binds to a target. Alternativelyelectrostatic or ion-pairing or salt-bridge interactions can bind to atarget. The beads can be paramagnetic beads that are only magnetic inthe presence of an external magnetic field. Alternatively, the beads cancontain permanent magnets. The beads can be added to complex samplessuch as aerosols, liquids, bodily fluids, extracts, or food. After (orbefore) binding of a target material, such as DNA, the bead can becaptured by application of a magnetic field. Unbound or loosely boundmaterial is removed by washing with compatible buffers, which purifiesthe target from other, unwanted materials in the original sample. Beadscan be small (nm to um) and can bind high amounts of target. When thebeads are concentrated by magnetic force they can form bead beds of justnL-μL volumes, thus concentrating the target at the same time it ispurified. The purified and concentrated targets can be convenientlytransported, denatured, lysed or analyzed while on-bead, or eluted offthe bead for further sample preparation, or analysis.

Separations are widely used for many applications including thedetection of microorganisms in food, bodily fluids, and other matrices.Paramagnetic beads can be mixed and manipulated easily, and areadaptable to microscale and microfluidic applications. This technologyprovides an excellent solution to the macroscale-to-microscaleinterface: beads can purify samples at the macroscale and thenconcentrate to the nanoscale (100's of nL) for introduction intomicrofluidic or nanofluidic platforms. Magnetic separations can be usedas an upstream purification step before real-time PCR,electrochemiluminescence, magnetic force discrimination,magnetophoretic, capillary electrophoresis, field-flow separations, orother separation methods well known to one skilled in the art.

The devices of the invention can accommodate the use of magnetic beads.For example, beads or bead slurry can be supplied to a port of acartridge. The beads can be mixed or suspended in solution within thecartridge using pumping, magnetic fields, or external mixers. The beadscan then be pumped to desired chambers or reservoirs within themicrofluidic device or cartridge. Beads can be captured within a chamberusing a magnetic field. Beads in a solution can be captured as thesolution travels through the magnetic field, or beads can be captured ina stagnant solution.

To illustrate methods of use of the cartridge, several examples arecontemplated. The first example is processing of nucleic acid from abuccal swab with paramagnetic beads to purify the sample followed by PCRamplification and bead purification of the PCR products. A secondexample describes performing immunomagnetic separations to purify cells,proteins, or other antigenic material using a binding moiety coupled tobeads. A third example describes performing molecular biology to preparesamples for sequencing technologies such as sequencing by synthesis,sequencing by hybridization, or sequencing by ligation. It would beknown to one skilled in the art that many different chemistries andbiochemistries can be used with the instant invention. These include,but are not limited to, enzymatic reactions, purifications on gels,monoliths, beads, packed beds, surface reactions, molecular biology, andother chemical and biochemical reactions.

The cartridge with integrated microchip can be formed of any cartridgeand microchip described herein. For example, the cartridge and microchipshown in FIG. 3, FIG. 4, and FIG. 5. A movable magnet (300) can bepositioned adjacent to the cartridge. The movable magnet can be moved byan actuator (310). The movable magnet can be used to apply a magneticfield within the cartridge or the microchip. In some embodiments, themovable magnet can be used to facilitate gathering or collecting ofbeads against a wall of a chamber within the cartridge or the microchip.

II. Reaction Module

The reaction module typically will comprise a reaction chamberfluidically connected to a microfluidic channel and the samplepreparation chamber through which the analyte passes. The reactionmodule can be adapted to place the analyte in a volume, e.g. anon-microfluidic volume, that is smaller than the original samplevolume. For example, the reaction module can comprise a chamber incommunication with a magnetic force that is adapted to immobilizemagnetically responsive particles on which the analyte is captured. Thebead bed typically has a volume that is smaller than the original samplevolume. The analyte can be released from to particles in the reactionchamber and a biochemical reaction, such as PCR, can be performed on theanalyte. The liquid volume in reaction chamber can be a non-microfluidicvolume e.g. between 10 and 50 microliters. Accordingly, the use ofanalyte capture materials that are flowable and immobilizable allowscapture, transfer and concentration of the analyte.

As shown in FIG. 6, temperature modulator can be fluidically connectedto the cartridge and microchip through reaction channel (250). Thereaction chamber (250) can be connected at an end (251) to thecartridge. The temperature modulator can be used for thermal cycling thetemperature of a reaction channel (250) containing a reaction mixtureand a nucleic acid enriched from a sample (collectively referred to asthe PCR reaction sample). A control mechanism can be used forcontrolling the operation of the temperature modulator. An opticalassembly can be used to monitor or control the reaction. The opticalassembly can introduce or detect light. For example, an optical assembly410 can be used for performing Real-time PCR or other real-time or endpoint measurements. In certain embodiments the temperature modulatoremploys a thermo-coupled Peltier thermoelectric module, a conventionalthermoelectric module, hot air, infrared light or microwave. In oneembodiment the temperature modulator uses a Peltier thermoelectricmodule external to the reaction channel to heat and cool the PCRreaction sample as desired. The heating and cooling of thethermoelectric module can be distributed over a region 350. Additionalviews of the temperature modulator 400 are shown in FIG. 7 and FIG. 8.FIG. 7 shows the reaction channel 250 in contact with a temperaturecontrolled region 350. The temperature modulator can also include amovable magnet 320 that is positioned by an actuator 330. The movablemagnet can be used to capture magnetic particles at position 340, asshown in FIG. 6. In some embodiments of the invention, the temperaturecontrolled region comprises two parts. The two parts can be parts of aclamshell that are clamped, locked, or held together to maintain thermalcontact with the reaction channel 250. One portion of the temperaturecontrolled region, portion 711 of FIG. 8, can be hinged to the secondportion of the temperature controlled region. The temperature controlledregions can have grooved channels for positioning of one or morereaction channels, as shown on the right side of FIG. 7 and in FIG. 8.The left side of FIG. 7 shows the temperature controlled region in aclosed configuration. Additionally, the temperature controlled regioncan comprise one or more constriction components, shown as 709 and 701in FIG. 8. The constricting points can pinch the reaction channel suchthat a portion of the reaction channel is isolated from another portionof the reaction channel. In some embodiments of the invention, thereaction channel is pinched in two locations such that a body of fluid,such as a reaction mixture, is isolated. Constriction components 709 and701 can mate with additional constriction components 707 and 705 tofacilitate pinching of the reaction channel.

Alternatively the temperature modulator can constrict the reactiontubing using a pinch clamp, as shown in FIG. 51. Use of the pinch clamp,which can be formed of a plastic such as Ultem, can reduce heat transferto the reaction channel. The reduction in heat transfer can reduce thelikelihood that the reaction channel has for being welded closed duringthermocycling or temperature regulation. Alternatively, differentmaterial tubing can be used as the reaction channel to ensure that thereaction channel can maintains its shape before and after thethermocycling or temperature regulation process. Different materialtubing can also be used to reduce rate of evaporation during thetemperature modulating process. Example materials include ethylvinylacetate, silicone, and silanized c-flex tubing.

The temperature modulating device can modulate temperatures at a rate of0.5 to over 3 degrees Celsius per second. The heater can utilize about25 to 100 Watts and a fan, which can be used to cool the temperaturemodulating device, can produce an air flow rate of at least about 75,100, 130, 150, 200, 250, or 300 cfm.

In one embodiment a sample preparation device comprising a cartridgeintegrated with a microfluidic microchip, which can be used to controlthe movement of fluid in the cartridge, can be used in conjunction witha temperature modulator 400 as a flow-through PCR thermal cycler.Driving force for moving the fluid can be an external pressure source oran internal pressure source, such as a MOVe valves within the microchip.A flow-through PCR thermal cycler can be used when highly sensitive orhigh throughput PCR is desired. There are many situations in which onemight want to sample air, blood, water, saliva, a cellular sample, orother medium in a sensitive PCR assay. This can be used to detect avariety of biological contaminants including influenza, bacterialpathogens, and any number of viral or bacterial pathogens. Flow-throughPCR can allow PCR to be practiced in an automated manner without theneed for human interaction. A flow-through PCR system can also serve asan early warning system in HVAC systems of buildings, airplanes, busses,and other vehicles, and can be used in the monitoring of blood, water,or other sample sources for the presence of an infectious agent or acontaminant.

As shown in FIG. 6, the flow-through PCR device takes a sample from acollection device, such as a buccal swab, a syringe, an air sampler,fluid sampler or other sampler and delivers it to a sample preparationdevice 1 (FIG. 6 is not necessarily drawn to scale). The sample isprepared in the preparation device 1, which in some embodiments mayinclude cell lysis, DNA, RNA, or micro RNA enrichment or purification,filtration, or reverse transcription. In one embodiment at least onenucleic acid is enriched. In another embodiment at least one enrichednucleic acid is prepared for PCR by adding the nucleic acid to PCRreagents (such as at least one DNA polymerase, RNA polymerase, dNTPs,buffer or a salt) and primers, (such as assay-specific primers orbroadly applicable primer sets for multiple target pathogens). Theseprimers may be chosen to selectively amplify at least one nucleic acidisolated from a specific pathogen (such as a mold, virus, bacteria,parasite or amoeba), gene, other desired nucleic acid, or anycombination thereof. The composition comprising at least one nucleicacid enriched from a sample, PCR reagents and primers is called a PCRreaction sample. In one embodiment, the flowthrough PCR can be used as acontinuous flow device while in other embodiments samples are moved intothe thermal cycling region and stopped.

The PCR reaction sample then flows through a reaction channel (250) to atemperature controlled device or region (350). In some embodiments thereaction channel is clear or transparent. In another embodiment thereaction channel is opaque. In one embodiment the reaction channel is acylinder. In another embodiment the reaction channel's cross sectioncomprises one or more planes forming a shape such as a triangle, square,rectangle, pentagon, hexagon, heptagon, octagon, nonagon, decagon, orother polygon. In one embodiment the volume of PCR reaction sample issuch that it takes up a small discrete length of space in the reactionchannel, the rest of which is occupied by air, gas, or a non-reactiveliquid, such as mineral oil. Air, gas, or a non-reactive liquid can beused to separate individual PCR reaction samples from each other. In oneembodiment the temperature controlled region (350) is thermallymodulated by one or more modules, including but not limited tothermo-coupled Peltier thermoelectric module, a conventionalthermoelectric module, hot air, microwave, or infrared light. In oneembodiment the thermal cycler uses Peltier thermoelectric modulesexternal to the tube to heat and cool the sample as desired. In oneembodiment a detection module (410) measures fluorescence, luminescence,absorbance or other optical properties to detect a signal emitted from aPCR reaction sample while it is located with a temperature controlregion, or after it has left a temperature control region. A detectionmodule can comprise a light source (such as a coherent light source orincoherent light source) used to excite a fluorescent dye (such as anintercalating dye, including but not limited to ethidium bromide orSyber green) in a PCR reaction sample, and the excitation light issensed with a photodetector (such as a CCD, CMOS, PMT, or other opticaldetector). Detection electronics can evaluate the signal sent from thedetection module (410).

In one embodiment, after the desired number of thermal cycles arecomplete, the PCR reaction sample is pumped or pushed further down thereaction channel, using pressure or vacuum, exiting the temperaturecontrolled region and passing into a second microfluidic microchip(500). The second microchip (500) can be attached at end (252) to thereaction channel (250). Microfluidic microchip (500) can comprisemicrovalves (510, 520, 530, and 545). Any three microvalves such as 510,520, and 530 or 510, 520, and 545 can form a pump. Microchannels 505,515, 525, and 540 can connect the pumps on the microchip. Downstreamdevices 535 and 550 can be connected to the microchip. Flow of materialto devices (535 and 550) can be controlled by the microvalves, forexample, by keeping either valve 530 or 545 closed while pumping ormoving fluid. In one preferred embodiment, the downstream device areanalytical devices that can be used for performing electrophoresis, massspectroscopy, or other analytical techniques known to one skilled in theart.

III. Post-Reaction Module

In certain embodiments, after the biochemical reaction is complete, thereaction product is processed before analysis. The system can include apost-reaction module adapted to process the reaction product. Thepost-reaction module can comprise a microfluidic device comprisingon-device valves and pumps adapted to route a fluid comprising thereaction product through a microfluidic channel into a processingchamber e.g., a non-microfluidic chamber. For example, if thebiochemical reaction is DNA amplification, e.g., PCR, and analysisinvolves e.g. capillary electrophoresis, processing can involveadjusting the salt concentration of the product containing volume. Thiscan involve for example, diluting the reaction product volume.

In another embodiment multiple reaction channels may be used in parallelto increase sample throughput. In yet another embodiment the system mayalert the user when amplification has occurred (a positive result),indicating that the target sequence is present. In one embodiment areaction channel is used for a single use only, then disposed of. In analternative embodiment a reaction channels can be used to amplify anddetect the presence or absence of PCR amplification products in multiplesamples. More than one PCR reaction samples can be loaded at intervalsand interspaced with a barrier bolus of gas or liquid to preventintermixing. In one embodiment samples are spaced apart in a manner sothat as one is undergoing thermal cycling another sample is in thedetection region undergoing interrogation. It will be obvious to oneskilled in the art that the PCR amplification can be replaced by othernucleic acid amplification technologies which may use thermal cycling orbe isothermal reactions.

In other embodiments, the device can perform isothermal reactions suchas sandwich assays using affinity reagents such as antibodies oraptamers to determine if cells, proteins, toxins, or other targets arepresent with the detection module (FIG. 6, 410) providing a reading ofthe amount of target present. In these applications, the cartridge 1 mayperform an affinity purification such as an IMS purification and thenadd a secondary antibody that may have a fluorescent label attached. Thesample can then move into region 350 where the thermal control is set tooptimize the reaction. Detection module (410) can then monitor thereaction. In one embodiment, a plurality of cartridges are ganged toreaction channel (250) and a series of boluses can be readout withdetector 410.

IV. Device for Product Analysis (e.g., Capillary Electrophoresis)

In one embodiment a complete sample-to-answer system is provide, whichcan comprise microfluidics, requiring coupling all steps together tomatch volumes and concentrations. Sample analysis using capillaryelectrophoresis is a standard analytical method that can be used withmicrofluidic sample preparation methods as described above. Capillaryelectrophoresis is readily adaptable to microfluidic microchips. In theinstant invention, capillary electrophoresis on microchips is combinedwith MOVe valves to provide control of samples, process beads toconcentrate the samples, and improve the loading and separations.

FIG. 48 show a sample source 6009 connected to a sample channel 6005,also referred to as a loading channel, that is mated with a separationchannel 6011. Two electrodes, 6003 and 6001, can be used to apply anelectric field to the separation channel. In some embodiments of theinvention, the sample source can pass through a MOVe pump in a microchipused to drive fluid flow within the sample channel. The sample channelcan be a microfluidic channel or an injection tubing. The injectiontubing can be flexible tubing or another flexible connector. Examples offlexible tubing include polytetrafluoroethylene tubing or silicontubing. The flexible connector can also connect to another cartridgeinterfaced with a microchip. Alternatively, the flexible connector canreturn to the cartridge that it originated from. The separation channelcan be a microfluidic channel, capillary tubing, or capillaryelectrophoresis tubing. The capillary tubing can have an outer diameterof about 150 to 500 microns and an inner diameter of about 10 to 100microns. The capillary can be polyimide or polytetrafluoroethylene clad.The capillary can be about 2 to 100 cm long. The capillary can be matedto the injection tubing or flexible tubing by first drilling a hole intothe injection tubing and then inserting the capillary into the flexibletubing. Alternatively, the capillary can be inserted into the flexibletubing without having to pre-drill the flexible tubing.

One of the two electrodes, for example electrode 6003, can be a cathodeand the other electrode, for example 6001, can be an anode. The cathodecan be any cathode, such as a forked cathode, described herein. Theanode can be connected to the separation channel using any devices knownto those skilled in the art. For example, the separation channel can bejoined to a reservoir by an Upchurch fitting, which is in electricalcontact with the anode, which can be a metallic electrode.

In some embodiments of the invention, a stabilizing component, shown atthe intersection of a separation capillary and injection tubing in FIG.49, can be used to align, seal, and/or protect the connection betweenthe separation capillary and the injection tubing. In some embodimentsof the invention, multiple injection tubings are aligned with multipleseparation capillaries using a stabilizing component. As shown in FIG.50, the stabilizing component can hold four injection tubings, shown asthe vertical tubings in the figure, and stabilize the connection withfour separation capillaries (not shown).

Panels 1-6 of FIG. 48 show a process for injecting a sample into aseparation channel. In panel 1, no sample is present in the samplechannel 6005. In panel 2, sample entering the sample channel from thesample source (6009) is shown. As sample is moved down the samplechannel, the sample intersects the separation capillary, as shown inpanel 3. The sample can be isolated by boluses of gas upstream anddownstream to the sample. Once sample is adjacent to the separationchannel, an electric field, which can be between 25 and 500 V/cm, isapplied between a first electrode 6003, which can be a cathode or aforked cathode, and a second electrode 6001, which can be an anode.Electrophoresis buffer, shown entering into the sample channel from thesample source, can also enter the sample channel, as shown in panel 3.The voltage potential and/or current between the anode and cathode candrop when an air bolus passes by the junction between the sample channeland the separation channel, reducing or preventing the injection of airinto the separation channel. The voltage potential and/or current dropcan be detected to ascertain when the sample and/or electrophoresisbuffer is adjacent to the separation channel. Once the electrophoresisbuffer is adjacent to the separation channel, as shown in panel 5, thecurrent and/or voltage drop between the anode and cathode can beincreased. This can allow for the separation of the analyte in theseparation channel, as shown in panel 6, as the electrophoresis bufferprovides ions for a high performance separation.

In one embodiment for STR analysis, the injection process is as follows:

-   -   A. The microfluidic channels can be filled with buffer.    -   B. The separation channel can be filled with gel while buffer is        pulled across the sample channel, thus sweeping the separation        polymer from the cross section formed by the separation and        sample channels.    -   C. The STR amplified sample (desalted and captured on beads) can        be captured on microchip 500, eluted in a low conductivity fluid        (water) containing the size standard, and pumped into the sample        channel with MOVe technology.    -   D. A field can be applied across the cathode and anode, with        “pull back” voltage on the sample and waste arms, to drive the        sample into the separation channel where it stacks at the head        of the separation polymer. As the sample is injected the        conductivity of the sample channel can quickly equilibrate with        the buffer in the cathode arms providing a single step        injection.

The forked electrode or cathode can be two metallic conductors, as shownin FIG. 46. The fluid path for a sample to be analyzed, as shown in FIG.46, can be along a loading channel. When the location of the sample isadjacent to the separation channel, the forked electrode can be used toinject the sample into the separation channel, as described herein. Theconductance of the material in the sample channel can be lower than theconductance of the material in the separation channel, which can be aseparation polymer. The difference in conductance can cause samplestacking when an electric field is applied through the forked electrode,which can be a cathode, and a downstream electrode, which can be ananode. The polarity of the forked electrode and the downstream electrodecan be reversed such that the forked cathode is the anode and thedownstream electrode is the cathode.

In some embodiments of the invention, an additional electrode can beused to reduce injection of gas into the separation channel or formationof bubbles within the sample loading channel which can lead to loss ofthe applied field on the separation channel. Injection of gas into theseparation channel or formation of bubbles within the sample loadingchannel can cause inconsistent separation of analytes and can bedetected by inconsistent current between the anode and cathode used toapply an electric field to the separation channel. Use of an additionalelectrode to circumvent or reduce injection of gas or bubbles into theseparation channel is shown in FIG. 47. The additional electrode can bea single wire run electrode or a cannular run electrode. The increasedsurface area and/or larger internal diameter of the cannular runelectrode can allow for a significant reduction in bubble formation orblockage and/or injection into the separation channel. In someembodiments of the invention, the cannula used for the cannular runelectrode and has an inner diameter of at least about 1/64, 1/32, 1/16,⅛, or ¼ inches.

In another embodiment shown in FIGS. 96A and B, the three-prongelectrode arrangement can be used to pre-concentrate analytes beforeinjection. In this method, the outside electrodes are given a charge thesame as that of the analyte, and the electrode between them and oppositethe electrophoresis capillary is given a charge opposite that of theanalyte. For example, in the case of nucleic acids, which are negativelycharged, the outside electrodes are given a negative charge and themiddle electrode is given a positive charge. This causes the analyte tobecome concentrated between the flanking electrodes and toward themiddle electrode. Then, the charge on the middle electrode is reversedand a voltage is placed across the electrophoresis capillary. This movesthe analytes through the capillary tube. The shown electrodeconfiguration for a capillary electrophoresis injector may include aforked electrode and another electrode. Both configurations showninclude a forked electrode having two points of contact located distallyto the connection with the electrophoresis capillary one electrodedirectly across from the lumen of the capillary. In one embodiment, thesecond electrode is a third fork in the forked electrode. In a secondembodiment the second electrode is not on the same circuit as the otherelectrodes. This allows two circuit paths to be employed at theinjector. In one method of using the three-electrode arrangement, thesample is moved into place opposite the capillary using the fluidicsystem. Then, the sample is injected into the capillary using only themiddle electrode which is opposite the capillary. Then, the middleelectrode is turned off, the flanking electrodes are turned on and areused to electrophorese the analytes through the capillary.

This invention provides a device for regulating temperature ofelectrophoresis capillaries, for example an array of capillaries. Anembodiment is shown in FIG. 98B. The electrically insulating circuitboard has a generally S-shaped path for placement of capillaries. Thegenerally S-shaped path is broken up into 6 different sections, 12, 14,16, 18, 20, and 22. These 6 different sections, 12, 14, 16, 18, 20, and22 separately regulate the temperature in the portion of a capillary inthermal contact with the particular section. Each of the differentsections, 12, 14, 16, 18, 20, and 22 is filled with an electrical paththat runs back and forth, e.g. in a serpentine shape in that section'sarea to fill that section's area. This electrical path that runs backand forth is shown in detail in section 22. Although not shown forpurposes of clarity in the illustration, the other sections 12, 14, 16,18, and 20 also are filled with an electrical path that runs back andforth in that section's area to fill that section's area.

The circuit board also has a row of apertures 10 that run along bothsides of the generally S-shaped path for placement of capillaries. Theapertures reduce heat transfer between the generally S-shaped path ofthe circuit board, and a remainder of the circuit board. Because air isa good thermal insulator, heat transfer is reduced between there twoparts of the circuit board. The circuit board itself is also a poorthermal conductor. In another embodiment, instead of rows of apertures,poor thermal conductive material is positioned between these two partsof the circuit board. Such reduction of heat transfer eases thermalregulation of the generally S-shaped path and the capillaries placed onthe generally S-shaped path. The apertures serve to reduce the thermalmass of the thermally regulated region to substantially the generallyS-shaped path and the capillaries placed on the generally S-shaped path.With less thermal mass, a desired temperature is reached more quicklyfor the generally S-shaped path and the capillaries placed on thegenerally S-shaped path.

The circuit board also includes an aperture 8 along the generallyS-shaped path toward the exiting end of the generally S-shaped path.Because of the absence of circuit board material, the aperture 8facilitates optical interaction with a capillary which is placed overthe aperture 8. The aperture 8 allows for fluorescence excitation anddetection using an optical configuration such as epi-fluorescent, andvarious skew illumination schemes.

The electrical path in various embodiments is a patterned, or etched,conductive trace bonded onto the electrically insulating circuit board.The patterned electrical path may be defined by “subtractive” patterningthat removes unwanted conductive material to leave the desiredconductive paths, or by “additive” patterning that adds additionalconductive material to form the desired conductive paths. The circuitboard may have the conductive paths on a single layer circuit board oras part of a multi-layer circuit board.

Various examples of conductive material in the electrical path aremetallic material such as copper, aluminum, silver, or nonmetallicconductive material such as graphite, or conductive ink, but may be anyother conductive material.

In contrast with the conductive material of the electrical path, thecircuit board material is nonconductive, commonly a dielectric material.

Each electrical path creates and defines a thermal area. In oneexemplary embodiment, six heating areas, each comprised of approximately1 m of 150 um wide copper traces that is folded into the shape needed togenerate the heater shapes shown below. Various embodiments vary thelength of the trace to shorter or longer than 1 m, depending on a lengthadequate for electrophoretic separation of analytes. Various embodimentswiden or narrow the width of the electrical paths, depending on anadequate resistance of the electrical paths to generate adequate heatfor thermal regulation of the thermally coupled capillaries. Variousembodiments increase or decrease the number of heating areas.

In some embodiments, an electrical path such as a trace has a width inthe range between 0.0001 to 0.5 inches, and a length in the rangebetween 0.25 to 750 inches.

Performing electrophoresis in a capillary allows the heat to beeffectively dissipated through the capillary walls. This allows highvoltages to be used to achieve rapid separations.

FIG. 2 is a top view of a thermal assembly, with a circuit board,electrical paths on the circuit board, a bundle of capillaries, andtemperature sensors.

On a circuit board such as the circuit board shown in FIG. 98A,electrophoresis capillaries are attached to the generally S-shaped path,such as by adhesive material. In the shown embodiment, a bundle of 8capillaries are attached. Other embodiments have any other number ofcapillaries ranging from 1 to a higher number, depending on a particularelectrophoresis application's requirements for parallel processing ofanalytes. The entering end 54 of the capillaries have fanned out ends,to facilitate injection of analytes into the different capillaries. Theexiting end 56 of the capillaries remains bundled together in thefigure.

In each of the separately thermally regulated areas or sections of thegenerally S-shaped path, a temperature sensor is in thermal contact. Thetemperature sensors shown are 32, 34, 36, 38, 40, and 42. Temperaturesensor 42 is in thermal contact not with the capillaries, but thecircuit board itself, or alternatively the ambient air. Examples oftemperature sensors are thermistors or other temperature-varyingresistance, or thermocouples or other temperature-varying voltagesource. In another embodiment, the temperature data of the separatelythermally regulated sections is not gathered by discrete temperaturesensor, but by the electrical paths themselves such as by theresistances of the electrical paths.

In the shown embodiment, temperature sensors are thermistors that areattached to traces that terminate on a portion of the circuit boardoutside of the array of thermal insulation apertures. The thermistorsare folded down across the capillary array and embedded in the adhesivethat bonds the capillary array to the board, to ensure good thermalcontact between the thermistors and the capillaries, while minimizingthermal loss from the heaters.

The temperature data generated by such temperature sensors help tothermally regulate the temperature of the capillaries in thermal contactwith the electrical paths. Electrical current through the electricalpath deposits thermal energy in the electrical path via Joule heating.The amount of deposited thermal energy varies with the amount ofelectrical current and resistance of the electrical paths.

In one embodiment, the invention provides a portable eight-channelintegrated automated sample-to-answer DNA forensics device. Such adevice accepts buccal swab samples or blood samples; performs STRanalysis; and outputs CODIS file ready for interrogation against adatabase.

In another embodiment, the invention provides an automated analysis forshort term repeats in a sample, wherein the method comprises extractingand purifying a sample comprising an analyte comprising a short termrepeat nucleic acid sequence; amplifying the short term repeat nucleicacid sequence; purifying the short term repeat nucleic acid sequence;and performing capillary electrophoresis on purified short term repeatnucleic acid sequence to determine the size of the short term repeatnucleic acid sequence.

Using standard technologies, the process from biological sample (e.g.,cheek swab) to data output of a CODIS file requires more than six hours.This includes DNA extraction, qPCR, DNA normalization, STR amplificationwith labeled primers, post-reaction sample preparation, CE analysis anddata analysis. The system of the present invention can perform thisentire process in less than 5 hours, less than 4 hours less than 3 hoursor even less than 2 hours.

In another embodiment, the invention provides an automated analysis forshort term repeats wherein the success rate of getting the correct STRfrom a raw or otherwise unpurified sample is at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, or at least 98%.

A. Detector

In one aspect, this invention provides an optical system for detectinganalytes in an array of capillaries, e.g. an array of 4, an array of 8or an array of up to 12 capillaries. In this system, the objective lensmoves in relation to the capillaries, the excitation source, and thecollection optics. As the objective moves, the excitation light passesthrough a different part of the lens and is focused at a different pointin the region of the capillaries. Movement of the objective results intranslation of the point of focus so that, so that each capillary can,in turn, be scanned.

In FIG. 99, the excitation source of the excitation beam 170 is a solidstate laser, the output of which is projected into the capillary 174using a beam combiner 162 placed at a 45 degree angle in the opticalpath immediately above the objective 160. In various embodiments thebeam combiner comprises a wavelength sensitive reflector or a spatialbeam splitter such as a small reflective dot placed on a transparentsheet of glass. The beam combiner is wavelength dependent, which iseasier to align than a spatial beam combiner.

The high numerical aperture objective is used both by the excitationbeam 170 on its way to the capillary 174, and by the optical signal ofemitted fluorescence from the capillary 174.

The optical signal of fluorescence emitted from the analytes of thecapillary 174 is collimated by the objective 160. The optical signalpasses through the wavelength sensitive reflector 162 and impinges on along pass filter 164 that rejects the portion of the optical signalincluding the excitation beam 170.

The fluorescence detection scheme is prism spectrometer based. Theoptical signal is then projected onto a dispersive prism 166, whichserves to change the angle of the rays according to wavelength. Thisdispersed optical signal is then focused on the plane of the detector170 using an image forming lens 168, causing different wavelengths ofthe dispersed optical signal to focus at different locations in theplane of the detector 170. An example of the detector 170 is a CCDcamera. An alternative is a CMOS camera or other optical sensors.

With an array that comprises 30 um diameter capillaries, the scan rangefor the detection device covers +/−0.8 mm for an eight channel array.This limited scan range minimizes the number of moving parts. Otherembodiments widen or narrow the scan range to accommodate a differentnumber of capillaries and/or different number of capillaries. As onlythe objective 160 moves, the excitation laser beam 170 remains veryclose to the center of the objective 160, even when the beam 170 islocated at the top of the end capillary in the array. The excitationbeam 170 impinges on the capillaries at different angles depending onthe location of the capillary in the array.

V. Integrated Analysis System with Disposable Cartridge

The invention provides for an integrated analysis system that caninterface with a disposable cartridge that integrates several functionsof the system, including sample extraction, thermal cycling, andpost-amplification processing. The integrated analysis system anddisposable cartridge can be used to analyze a sample obtained from asource and provide information regarding the source. For example, thesystem and cartridge can be used to extract nucleic acid from aplurality of samples and analyze the extracted nucleic acids for shorttandem repeats to obtain genetic information regarding the source.

An overview of the integrated analysis system and disposable cartridgeis shown in FIG. 63. The integrated analysis system can includehardware, consumables, software, and documentation. As shown in FIG. 64,the hardware components of the integrated analysis system can includeone or more enclosures, electronics, pneumatics, optics, thermocyclers,electrophoresis systems, and cartridge covers. Additional components ofthe hardware are shown in FIG. 64. The integrated analysis system canalso include one or more control systems, power systems, computers, anddisplays. As shown in FIG. 65, the software components of the integratedanalysis system can include software providing for a graphical userinterface, data analysis, a core, and scripts. The core can be used forthe control of the hardware. The scripts can be programs that are run.The programs can correspond to particular analytical procedures.

As shown in FIG. 66, the consumables can include one or more capillaryarrays, reagent cards, cartridges, and separation polymer cartridges.The reagent cards can include reagents for performing analyticalreactions. The cartridge can include chambers for receiving samples andchips having one or more pneumatically actuated valves for controllingfluid transport. Other consumable components are shown in FIG. 66.

FIG. 67 shows documentation that can be included with the integratedanalysis system. Documentation can include test documentation, manuals,project documentation, requirements and specifications, productdocumentation, and risk management.

The integrated analysis system and disposable cartridge can be enclosedin a container having a total volume of less than about 10 ft³, lessthan about 5 ft³, less than about 3 ft³, less than about 2 ft³, or up toabout 1 ft³, such as a Pelican model 1610 case. The pelican case #1610 @2.2 ft³ can be used to contain all system parts including user controland spare cartridges. The dimensions of the container can be up to aboutor about 22×17×11 inches. The container can have wheels and handles forfacilitating transportation of the system. The container can be paddedor reinforced such that the system is protected from damage duringtransportation or use. The container can be securely closed or have oneor more alarms to prevent and/or deter unintended entry and/or theft. Anexample of an encased and integrated analysis system is shown in FIG.68. Accordingly, this system incorporates into a suitcase-sizedcontainer instrumentation to accept a biological sample and produce ananalysis of the genetic content of that sample. The system ismultiplexed and can perform a plurality of operations at once, forexample, at least 2, at least 4, at least 6, at least 8, at least 10, oreven at least 12, or more samples in parallel. In a specific embodiment,the system can perform 2, 4, 6, or 8 samples in parallel.

The case load can be <50 kg and can be shipped without protection. Noadditional packaging may be required, enabling rapid deployment of thedevice. The lid can be opened and the device can be plugged in uponarrival. The small size and two man lift can enable setup and transferwithout need for lift equipment. In some embodiments of the invention,the system includes an internal power source or a power generator. Theinternal power source can be a battery for powering the system in theabsence of an electrical source. The battery can be a rechargeablebattery or can be a non-rechargeable battery. The power generator can bea fuel cell, a solar cell, or any other type of power generator known toone skilled in the art.

The container or case can also have one or more internal enclosures. Theinternal enclosures can protect hardware from damage or spills. Anexample of an internal enclosure is shown in FIG. 68. The internalenclosure has two vents with radial cutouts to allow for air-flow. FIG.69 shows the system with the internal enclosure (1.0, bottom right)removed from the container, exposing the hardware of the integratedanalysis system. FIG. 69 also indicates the location of the optics(4.0), pneumatics (3.0), cartridge (10.0), thermocyclers (5.0),cartridge cover (7.0), reagent card (9.0), and electronics (2.0).

Under operation, the system may draw hundreds of watts which may need tobe exhausted from the chassis. Two rotary fans can draw air through aset of filters and provide sufficient cooling to maintain the internalchassis air temperature within 5° C. of ambient with two fans and <10°C. with a single fan. Fans can be brushless type to increase life timeto >25 k-hr.

The air and temperature control subsystem manages thermal flow withinthe suitcase enclosure, the thermal cycler, and the CAE. The layoutemploys a forced-air, internal plenum with inputs at the sides of theApollo 200 System and outputs filtered air at the front and back of thecartridge to facilitate adequate thermal management and minimizeparticle introduction.

Another diagram of an enclosed integrated analysis system is shown inFIG. 70. FIG. 70 shows a capillary electrophoresis ring for performingseparations, a CCD-based detector for detecting analytes, an integratedcassette (combined disposable cartridge with reagent card), pneumaticsolenoids, cassette clamp and interface (combined pneumatic manifold andcartridge cover), embedded tablet PC with touch-screen interface,exhaust fan, pneumatic pressure source and ballast, and electronics.

The computer and control software can integrate the system and providethe electronics interfaces, timing and control. The computer can be anindustrial PC running Microsoft XP. Software can be Software controlsoftware. The main user interface can be a touch screen panel withauxiliary interface by monitor (VGA or higher res), external keyboard(USB) and mouse (USB) that may not included in the enclosure. A bar codereader (such as Keyence BL-180) and a GPS unit (such as an OEM modulebased on the Sirf-Star iii Chipset) can be connected via USB to thecomputer.

FIG. 71 shows a view of an enclosed integrated analysis system. FIG. 71shows a touch screen (1.5.1), barcode reader (1.5.2), GPS (1.5.3), case(1.1), power supplies (1.7), chassis and structure (1.2), Internalpartitions and baffles (1.3), electronics (2.1-2.5), separation andpolymer fill device (6.2), and separation polymer cartridge (11.0). Theelectronics include a communication controller (2.1), solenoid valvecontroller (2.2), temperature control (2.3), motor control (2.4), andsensor board (2.5).

FIG. 72 shows a close-up view of the optics and pneumatics systems ofthe enclosed and integrated analysis system. The optics include thealignment components and base (4.1), excitation source (4.2), detector(4.3). The pneumatics include the vacuum supply (3.1) and the pressuresupply (3.2).

Industrial grade power supplies can be used to improve the life time ofthe system. The rating of the power supplies may not be more than 500 W.Interconnects that meet vibration standards and are proven to meet longlife times can be used.

Chassis design for mounting subassemblies can include a number ofstructures which may utilize aluminum castings to reduce weight and costof high volume parts. Aluminum structures can support subsystems 2,3,4,5,6 and 11 (FIG. 64). Structures utilizing sheet metal parts cansupport subsystems 1.5 and 2. The design can allow for reduced weightand cost while maintaining rigidity and durability to meet vibrationsspecifications for transportation. Structures can include internalisolation between the enclosure and system/sub-systems.

Partitions to separate the user manipulated sections, such as thecartridge loading, from the electronics and optics to avoid spillage;and baffles to keep stray light from entering the optics can be madefrom plastic or aluminum parts.

The chassis can be designed with a cartridge-handling subsystem. TheCartridge-Handling Subsystem interfaces the chassis with theapplication-specific cartridge and is located at the front center of thechassis (FIG. 39). The Cartridge-handling subsystem can be activatedwhen the user pushes the soft button ‘Load.’ The Cartridge-handlingsubsystem can accept the cartridge from the user in a cradle similarlyto a VCR mechanism which can then be actuated to move the cartridge intothe chassis.

The cartridge will first move away from the user and then down. A topplate can seal the top of the cradle. Fluidic and pneumatic connectionscan be through the top and bottom of the cartridge through the top plateand a bottom plate both under spring force. All connections can beprotected from the environment by the top plate during processing andwhen stored. The Cartridge-Handling Subsystem and the first cartridgeare the highest risk items in the project.

As the Cartridge-Handling Subsystem completes the cartridge input, boththe pneumatics and fluidics are sealed as the cartridge under the springpressure seats onto miniature ‘O-ring’ seals on the bottom plate.

The unit can be constructed to meet standards in transportationvibrations and environmental extreme conditions. The unit can beutilized in a variety of case orientations. The unit can be designed tofunction at extreme temperatures and humidity specifications. The unitcan be designed to prevent light-leaks.

The components of the integrated analysis system and disposablecartridge are depicted in FIG. 73.

A reagent pneumatic assembly engages the cartridge and can perform anumber of functions. A cartridge cover connected to an air supply isshown on the top left portion of the schematic shown on FIG. 73. Adepiction of the cartridge cover is shown in FIG. 74. FIG. 74 shows theair supply ports for a vacuum source (3.4 Vac) and a pressure source(3.4 Press). The cartridge cover also includes a magnet assembly (7.1),reagent handling interface (7.4) and a heater assembly (7.2).

The Cartridge cover interfaces with the top of the Cartridge and theReagent card. It can include several functions: Magnet positioning formagnetic particle capture in the sample extraction process, Heaterpositioning for the sample extraction lysis process, connection of theCathode Tubing to feed diluted products to the Electrophoresis System,integration of pressure and vacuum to the Reagent Card, illumination forcontrol of the Sample Loading process and Magnetic Particlere-suspension before Reagent Card activation.

-   -   A. A primary functionality of the Cartridge Cover is to align        with the Cartridge, activate the Reagent Card and interface with        the Cartridge to provide extended functionality. The functions        can include the following, referring to FIG. 74.        -   A. Magnet assembly (7.1): After lysis the DNA from the            sample can be captured on magnetic particles and prepared            for further processing by washing to remove contaminants and            PCR inhibitors. To provide for the capture and re some or            lease of the particles within the Cartridge the Cartridge            Cover contains an assembly to facilitate the movement and            location of an array of rare earth magnets in respect to the            capture chamber on the Cartridge. This assembly consists of            a magnet array and a mechanism. Magnets can be moved into            and out of position to exert a magnetic force on            compartments in the cartridge that can contain paramagnetic            beads.        -   B. Heater assembly (7.2): Cell lysis is carried out at            elevated temperature, typically 60-80° C. The lysis takes            place within the sample chamber which is heated by the            Heater assembly. The Cartridge Cover positions the resistive            heater around the sample chamber and provides            interconnections to the Thermal management component of the            Electronics. Temperature feedback is supplied by            thermocouples in the heater.        -   C. Cathode Tubing Connection (7.3): After PCR and dilution            the sample, now diluted product, is moved to the Cathode            Tubing for injection onto the capillary. The Cathode Tubing            is connected through the Cartridge Cover and interfaced with            the Cartridge by a low-pressure seal such as a face seal or            ferrule type connection. This connection is leak free, clean            and low dead volume.        -   D. Reagent Handling (7.4): To maximize MOVe valve            performance it is sometimes useful to feed the valves with            pressurized reagents. Within the Cartridge Cover there are            several ports that, when the Reagent Card is activated            during system setup, pressurized specific reagent wells. In            the same manner, the best scavenging of waste materials is            often achieved with vacuum assist. Again, upon Reagent Card            activation, specific waste wells may be evacuated to            accomplish this. The interface with the Reagent Card is            through cannulae, mounted in the Cartridge Cover, that            pierce the top seal on the Reagent Card.        -   E. Sample Loading (7.5): To facilitate the proper and            controlled loading of samples, positive controls, negative            controls and allelic ladder the Cartridge Cover can be            fitted with sensors and illuminators that work in a closed            circuit fashion with the software to monitor and control the            activity. Depending on the Cartridge Type and run setup            parameters the system can “activate” one or more, e.g., from            five to all eight, of the Sample Chambers. A chamber can be            activated by illuminating the chamber with an LED or other            light source built into the Cartridge Cover. When a swab,            FTA punch or control is inserted into the chamber it is            detected by an optical sensor built into the Cartridge            Cover. The system then turns the illumination off,            effectively de-activating the chamber. If a sample is placed            into a de-activated chamber the system enters an error mode.            The illuminators and sensors are interfaced to the Sensor            Board in the Electronics.        -   F. Thermocycler Interface (7.6): The Reaction Tubing on the            Cartridge is mechanically aligned to the Thermocycler            components by the Cartridge Cover.        -   G. Magnetic Particle Re-suspension (7.7): Before the Reagent            Card is activated the Magnetic Particles can be re-suspended            as they may have settled during shipping and storage. The            Cartridge Cover can interface a re-suspension device, such            as an ultrasonic probe, peizo vibration probe or mixing            mechanism, to the Magnetic Particle well of the Reagent            Card.

The cartridge cover can interface with a cartridge, shown within thedashed lines below the cartridge cover on FIG. 73. The cartridge covercan apply pneumatic pressure to the cartridge and/or be configured toapply a magnetic field and/or heat to one or more components of thecartridge. A depiction of a cartridge interfaced with the cartridgecover and pneumatic manifold is shown in FIG. 75. FIG. 75 also shows afan and ducting (5.3) and thermocycling components: thermal plate (5.1),thermoelectric (5.2), and controls (5.4).

The cartridge, shown in dashed lines on FIG. 73, can include one or morechambers, fluidic connections, microchips, and/or pneumatically actuatedvalves. A top view of the cartridge is shown in FIG. 76, FIG. 77, FIG.78, and FIG. 79. FIG. 79 shows reagent distribution chambers, capturechambers, sample chambers, and post amplification chambers. Thecartridge can also have a receptacle for receiving reagent chambers thatare on a reagent card. The reagent card can have a variety of chambers,including reagent chambers (FIG. 76, 9.0), reaction chambers, and wastechambers. The reagent card (also referred to as a removable reagentcassette) can be manufactured separately from the cartridge. The reagentcard can be inserted or interfaced with a reagent card slot (shown inFIG. 79). The cartridge can be made of a piece of injection-moldedplastic. The volume of the chambers, for example the sample chambers,capture chambers, and post amplification chambers can be about or up toabout 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 5, or 7 mL. The cartridge can havedimensions of about or up to about 1, 2, 5, 7, 10, 15, or 20 cm in widthor height and about or up to about 0.2, 0.5, 0.75, 1, 2, 3, 4, or 5 cmin thickness.

The Cartridge element consists of several integrated parts. Theseinclude a piece that contains reaction chambers and that functions as afluidic manifold. The chambers, on one side of the piece, communicatewith the opposite side of the piece through ports. The opposite, orfluidic side, is engaged with fluidic chips comprising channels anddiaphragm valves and pumps that move liquids between reaction chambersand reaction tubing. The chips can be attached to the fluidic sidethrough a gasket. In certain embodiments, the cartridge comprises aplurality of chips. In this case, the fluidic manifold piece cancomprise channels that fluidically connect one chip with another. Suchchannels are depicted, for example, in FIG. 94. They can be grooves,troughs or other depressions in the surface of the piece that are sealedby a gasket between the underside of the piece and the microfluidicchips. The Reagent Delivery fluidics, Sample extractor fluidics, thePost-amplification fluidics, the top piece, the bottom piece and theReaction Tubing. The top piece, also referred to as the fluidicmanifold, is a plastic assembly and the fluidics are three layer glassMOVe chips that direct and control the flow of reagents through thesystem. The assembly connects to the pneumatic manifold and interfaceswith the Cartridge Cover. The Cartridge facilitates all of the sampleprocessing up to the loading of the amplified, labeled products onto theseparation column. The Cartridge contains all the waste it produces andis replaced after each run to avoid contamination.

The top piece can be a molded plastic part with chambers for the reagentfill, sample extraction particle capture, sample insertion/lysis andproduct dilution. It also can have a section of silicon tubing used forreactions as well as an interface section for connection to the cathodetubing and an area for interface with the reagent card. For example, thesilicon tubing can be used as a thermal cycling reaction chamber that isclamped at both ends during thermal cycling. The piece is made up ofseveral parts. The fluidics components are attached to the bottom of thetop piece with double sided adhesive.

The bottom piece can function as a gasket and seals of the channelsmolded into the Top Piece and provides a mounting surface andpassageways, called vias, to the MOVe fluidic components.

The reagent delivery fluidics can deliver a preset volume of reagent tothe Sample Extractor section facilitating sample processing. Thisdelivery relies on pressurization of the Reagent Card wells which feedsthe Reagent distribution fluidic MOVe device. The MOVe device controlsthe filling and disposition of the reagent chamber in the top piece.Pressurized air is also available for delivery to the Sample extractorsection of the Cartridge. The section is comprised of one or more MOVechips attached to the bottom of the integrated Cartridge bottom piece.The associated manifold and chamber features are connected to thedownstream sections through the top piece.

The sample extractor can include one or more syringe chambers that canbe configured to receive one or more samples, such as buccal samples orswabs. In FIG. 76, the syringe chambers are the row of verticalcylinders that are second from the left. The syringe chambers are alsoreferred to as capture chambers (shown in FIG. 79). The syringe can beheated by a component on the cartridge or by a component on thecartridge cover.

Within the Sample extractor the swab/brush/disc is rinsed, the cells arelysed, the DNA is captured on particles, the particles are cleaned anddelivered to the Reaction tubing for capture. The fluidics is a threelayer glass MOVe chip attached to the integrated Cartridge bottom piece.The MOVe device distributes the flow of reagents from the Reagentdistribution section to; the lysis/sample insertion chamber, the capturechamber or the Post-Amplification fluidics.

The Post Amplification fluidics interfaces with the Sample Extractorsection, the Dilution Chamber, the Reaction Tubing and the CathodeTubing. These fluidics consist of one of more three layer glass MOVedevices attached to the bottom piece. The Post Amplification fluidicscontrol the particle capture from the Sample Extractor section, meterand move premix from the Reagent card to the Reaction tubing, moveproduct from the reaction tubing to the dilution chamber, and metersdiluent from the Reagent card to the dilution chamber. It also metersand moves buffer, water and sample to the cathode tubing.

The cartridge can include reaction tubing (shown in FIG. 77). The tubescan be interfaced with a temperature controller, such as a thermocycler(shown in FIG. 73). The tubes can also be interfaced with a magnet(shown in FIG. 73).

The Thermocycler module temperature cycles the PCR-STR reactions in theReaction tubing. It has several distinct components. The Reaction tubingis enclosed in a two piece shell that provides thermal contact and, whenfully closed, pinch seals the silicon tubing. The bottom piece of theshell is part of the Cartridge. The top piece of the shell and thethermal control elements are aligned to the bottom piece and the tubingby the Cartridge cover. During particle capture and premix load theshell is in the “open” position; prior to cycling the shell is moved tothe “closed” position. In the closed position the top and bottom piecesare in contact with each other which can facilitate thermalcommunication and pinching of the tubing which limits reaction plugevaporation and movement. Heating and cooling of the assembly isprovided by a thermal electric device. The thermal control duringcycling is driven by a controller which is managed by the electronics.Feed back is provided by a thermocouple permanently bonding to the toppiece of the shell.

The thermal cycler temperature control can use Software objects and athermal control board updated to 12-channels. Heating of the BeadStormsample extraction portion of the DNA Profiling cartridge and of thecapillaries of the CAE cartridge can use resistive heating monitored bythermocouples. The STR reactions can use resistive heating and fancooling as the base case. Peltier/thermoelectric heating and cooling canalso be applied as needed for extreme temperature ranges; MBI usesthermoelectric with MOVe microchips in a project with a strategicpartner. The air from the thermal cycler can be output to the plenum.Control of temperature can be exerted through the MBI TemperatureControl Board controlled by existing Software.

The chambers can be fluidically connected to a cartridge manifold. Thecartridge manifold is shown in FIG. 80. The manifolds can include one ormore channels. These channels can be for swab extractor outputs, reagentdistribution, post amplification to thermocycler channels, lysischannels, ethanol channels, waste channels, and bead channels.

The cartridge manifold can have one or more channels that fluidicallyconnect the chambers on the top layer of the chip to one or moremicrochips. The cartridge manifold can also fluidically connect one ormore microchips to each other, allowing for a fluid in one microchip tobe transported to a second microchip. For example, material in thereagent chambers (shown in FIG. 73) can be transported to the swabsyringe (shown in FIG. 73) by transport through the followingcomponents: the reagent distribution manifold section, the reagentdistribution chip, the reagent distribution manifold section, the swabextractor manifold section, the swab extractor chip, and the swabextractor manifold section.

Microchips are shown as square outlines in FIG. 81 and FIG. 82, which isa view of the base of a cartridge. The chips can be post amplificationchips, sample extractor chips, and reagent distribution chips. Thechips, also referred to as microchips herein, can have one or morepneumatically actuated valves. The valves can be used to control fluidflow by blocking or opening fluidic passages or providing a drivingforce for fluid movement. The valves can be controlled by a pneumaticmanifold (shown in FIG. 73). The pneumatic manifold (shown in FIG. 73)can be similar to any other pneumatic manifold described herein. Thepneumatic manifold can be connected to an air and vacuum source.

The chips can be fluidically connected to each other by channels in thecartridge. The channels in the cartridge can be an alternative to tubesthat fluidically connected microchips, as described elsewhere herein.This can reduce the space required for the integrated analysis systemand the time required to perform an analysis reaction.

As shown in FIG. 73, the reagent card can include reagent chambers for,e.g., lysis, ethanol, bead, waste, syringe, water, ladder, premix,diluent, buffer, and waste chambers. The premix can be any premixdescribed herein. In some embodiments, the premix is a premix forperforming a nucleic acid amplification reaction. The reagent chamberscan have a volume of about or up to about 0.1, 0.2, 0.5, 0.75, 1, 2, 5,7, or 10 mL. The chambers can include features or mechanisms for keepingreagents cooled, heated, or mixed. For example, the chambers can includeagitators or mixers. The reagent chambers can be sealed prior toengaging the cartridge. Engaging the reagent card with the cartridge canreversibly or irreversibly place the reagent chambers in fluidicconnection with the cartridge. In some embodiments, the cartridge hasone or more cannulae that puncture a seal that encloses the reagentchamber. The seal can be a plastic seal or a rubber seal. In someembodiments of the invention, the rubber seal can effectively re-sealthe chamber after dis-engagement. Accordingly, the card can be engagedwith the cartridge in an inactive configuration. When the cartridge isengaged with a pneumatic manifold, the cover can depress the card intoan active position in which the cannulae puncture the reagent chamber onone side and the cover punctures the reagent chambers from another side.This places the reagent chambers in fluid communication with thereaction chamber through channels connected by the microfluidic chips.This also allows positive pressure to be exerted to present reagent tothe chips, which pump it to directed locations, as well as allowingvacuum to be exterted to pull waste presented by the chips into wastechambers on the card.

The card is designed with chambers to accommodate specific fill volumeswithout entrapping air. Both sides of the chambers are sealed with anelastomeric membrane. This eliminates the need for centrifugation beforeuse. The card is fixed to the Cartridge and locked into a raised “safe”position. This is how the card ships with the Cartridge. In thisposition the bottom seal is aligned to the cannula on the top of theCartridge. A safety mechanism assures that the card does not move to thelower “active” position where the cannulae on the Cartridge pierce thereagent cards seals.

The following are components of the reagent card, referring to FIG. 66.

-   -   A. Reagent Set (9.2): The Reagent Set and Reagent Card loading        process and equipment are loaded with reagents adapted for        performing the particular chemical reaction desired. For        example, they can include reagents for performing STR analysis        on one or more CODIS markers. The card can contain the following        reagents:    -   B. Diluent (9.2.1): The diluent is used to lower the salt        concentration of the PCR reaction product and introduce a size        standard. The size standard in the diluent can have fragments        from approximately 60 to 600 bp.    -   C. Wash Solution (9.2.2): The wash solution is used to clean the        magnet particles and facilitate transfer to the Reaction Tubing.    -   D. Lysis Buffer (9.2.3): The Lysis Buffer is used to lyse the        cells in the sample.    -   E. Allelic Ladder (9.2.4): The Allelic Ladder is included on one        type of Cartridge. The Allelic Ladder is used to establish a        standard position for all the possible Loci sizes. When used,        the Allelic Ladder takes the place of one channel, during the        sample loading process this channel may not be loaded with a        standard or sample.    -   F. Capture Particles (9.2.5): The Capture Particles are used to        facilitate the isolation, purification and volume reduction of        the sample DNA.    -   G. Run Buffer (9.2.6): The Run Buffer is used as an electrolyte        in the electrophoresis process.    -   H. Premix (9.2.7): The Premix supplies the primers, buffer and        enzyme to run the PCR reaction    -   I. Water (9.2.8): Water is used to prepare the Cathode Tubing        for the reaction products.

A single or set of small, quiet pumps can be used along with ballastsand precision regulators to supply the required pressure and vacuum. Theregulators can be factory set and monitored by the system throughelectronic pressure transducers to assure accurate and predictablepneumatic performance at all times. MOVe valves have very low flow butrequire well regulated pressure. The Cartridge Fluidics manifoldprovides primarily for MOVe valves. The Cartridge Cover/Reagentsmanifold supplies the pressure feed reagents and internal pressurizedair requirements of the cartridge as well as vacuum for the waste wells.This is slightly higher flow than the MOVe valves. The Mechanismsmanifold is higher flow but usually may not require the same accuracy ofregulation.

The pneumatic manifold provides for the mounting of the Cartridge andthe control interface to the MOVe fluidics. The manifold has 3-waysolenoids with a delivery port switching between vacuum and pressure.The solenoids are addressed by the Solenoid Valve Controller componentof the electronics sub-system. The solenoids are mounted in a manifoldthat distributes the pneumatics to ports that align with inputs on thebottom of the MOVe microfluidic chips that are part of the Cartridge.This manifold also serves as the alignment and interface component tothe cartridge.

The pneumatic manifold can be integrated into the chassis of theencasement such that the pneumatic manifold can be moved away fromunderneath the cartridge cover. This can facilitate loading of thecartridge into the integrated analysis system. FIG. 83, FIG. 84, andFIG. 85 show loading of a cartridge into an integrated an encasedanalytical system. FIG. 83 shows the pneumatic manifold in a positionthat is not underneath the cartridge cover. In FIG. 83, the cartridge ispositioned above the pneumatic manifold. FIG. 84 shows the cartridgeengaged with the pneumatic manifold. The pneumatic manifold can then bemoved or slid to a position such that the cartridge can be engaged withthe cartridge cover, as shown in FIG. 85. FIG. 69 shows the cartridge ina position such that the cartridge is engaged and fluidically connectedwith the cartridge cover and pneumatic manifold.

To maximize MOVe valve performance it is sometimes can be useful to feedthe valves with pressurized reagents. Within the Cartridge Cover thereare several ports that, when the Reagent Card is activated during systemsetup, pressurized specific reagent wells. In the same manner, the bestscavenging of waste materials is often achieved with vacuum assist.Again, upon Reagent Card activation, specific waste wells may beevacuated to accomplish this. This manifold is a mixed manifold of 3-waysolenoids delivering either pressure or vacuum to the output port. Thethird port is generally vented to atmosphere. The solenoids interfacewith the Solenoid Valve Controller component of the electronics.

Pneumatic cylinders are used to move and set a variety of mechanismswithin the system. These include the Thermocycler, Cartridge Cover andcomponents within the cover. The mechanism manifold is pressure only,utilizing 3-way solenoids to deliver pressure to the output port andvent to atmosphere with the third port. The solenoids interface with theSolenoid Valve Controller component of the electronics.

The cartridge cover, cartridge, and pneumatic manifold can be used toextract nucleic acids and perform amplification reactions, as describedelsewhere herein. Amplified nucleic acids can be delivered to one ormore electrophoresis channels (shown in FIG. 73) through fluidicconnectors (shown in FIG. 73). The electrophoresis channels can beelectrically connected to a cathode and anode. The cathode and anode canbe electrically connected to a high voltage controller (shown in FIG.73). The electrophoresis channels can be filled with separation polymerusing the separation polymer module (shown in FIG. 73).

The Electrophoresis hardware consists of the High Voltage Supply andControl, the Separation Polymer Fill Device and the Capillary ArrayThermal Control. It also facilitates the mounting of the Capillaryarray. The thermal control provides precise heating of the capillariesduring electrophoresis.

The cathode is part of the Capillary array and held in place by theElectrophoresis System hardware. The anode is part of the SeparationPolymer Fill Module and is connected to the Electrophoresis Systemhardware. The high voltage control is an MBI HV board switching an OEMhigh voltage source. This action is managed and monitored by theelectronics. High voltage is supplied to the anode electrode whilesixteen cathode electrodes (two per channel) are held at ground. Currentmonitoring is done between the cathodes and ground and is monitored forindividual channels on the MBI HV board. The cathode electrodes are partof the Capillary Array and connections are made to the HV system duringarray installation. The anode electrode is part Separation polymer fillmodule and the electrical connection to the cathode is through electrodeimmersion in the separation polymer.

The Separation polymer fill provides separation polymer to thecapillaries through an interface at the anode. It connects to theElectrophoresis System hardware for anode high voltage supply andcontrol. The Separation Polymer Fill Cartridge attaches to the moduleand supplies pressure and reagent for every run. The system is pressuredriven and controlled by solenoids managed by the electronics. Pressurecan be monitored by electronic transducers monitored by the Electronics.Safety can be provided by passive and active pressure relief paths.

The Separation Polymer Cartridge contains the separation polymer, apressure canister, and a waste receptacle. The assembly is installed inthe system before the run. During the run the system uses the pressureto flush the anode and refill it with fresh polymer then forces thepolymer into the capillary array.

The high voltages used in capillary array electrophoresis (CAE) producesubstantial thermal loads that can be managed to eliminate thelikelihood of thermal runaway, due to Joule heating, disturbing theelectrophoretic process. CAE is generally run at elevated temperature,40-80° C., producing a requirement of uniform thermal characteristics ofthe capillaries during the heat producing electrophoresis process.Affective transfer of Joule heating is required along with thermaluniformity to produce predictable, high resolution electropherograms.The heating and physical thermal management of the capillaries residesin the Capillary Array assembly. The interface and control of thetemperature is through the Capillary Thermal Control component of theElectrophoresis System and the Temperature Control component of theElectronics.

The Capillary array includes the anode end connection, cathode tubingand electrical connections, window holder, heater, holder and eightcapillaries; it provides for the separation of the labeled products andinterfaces with the Electrophoresis System (6.0) and the Optics (4.0).The assembly is reusable and requires replacement by a trainedtechnician at a prescribed interval, likely during regular maintenance.Replacement includes removing the old assembly, connecting the newassembly and aligning the capillaries to the Optics assembly. The anodepiece interfaces the capillaries with the Separation Polymer Fill Device(6.2) and the anode electrode (within 6.1). It is also connected to awaste. The cathode tubing provides an interface between the capillariesand the Cartridge (10.0). It is also connected to a waste. Theflow-through TREKI injection system utilizes two cathode injectionelectrodes which are permanently fixed and removed with the assembly.The window holder places and holds the detection window in the opticalpath and interfaces with the alignment system on the Optics assembly.The eight fused quartz polyamide coated capillaries, when filled withthe separation polymer, facilitate the size mediated separation of thelabeled products. Windows in the polyamide allow for the excitation anddetection of the products as they move through the system. Capillarythermal control is by close contact to a material with high thermaltransfer. Temperature control of the assembly is by resistive elementand is driven by the Temperature Control electronics (2.3). Feed back isprovided by a thermocouple.

The high voltages used in capillary array electrophoresis (CAE) producesubstantial thermal loads that can be managed to eliminate thelikelihood of thermal runaway, due to Joule heating, disturbing theelectrophoretic process. CAE is generally run at elevated temperature,40-80° C., producing a requirement of uniform thermal characteristics ofthe capillaries during the heat producing electrophoresis process.Affective transfer of Joule heating is required along with thermaluniformity to produce predictable, high resolution electropherograms.The system can utilize materials with high thermal transfercharacteristics and good thermal uniformity such as aluminum andgraphite coupled with powerful, flexible resistive heaters and fineresolution thermal control and monitoring to maintain tight thermaltolerances.

A detector (shown in FIG. 73) can be used to observe or monitormaterials in the electrophoresis channels. The detector can be a CCDcamera-based system, or any other detection system described herein.

The Optical detector assembly detects the separated labeled products andsends the data to the software. The assembly consists of excitation andcollection optics, an excitation source, a prism and a camera. Theexcitation source is a DPSS laser which passes through specializedoptics which shape the beam into a line. The line is focused on thecapillary windows. The emission generated by the excitation of thefluors as they pass through the window area is collected by an objectiveand passed through a prism which separates the light into its componentwavelengths. The output of the prism is focused on a camera whichcollects data in which the capillary array is in one axis and theemission spectra is in the other. Based on off the shelve reagent kits,the emission spectra can initially be separated into five bands. Thenumber of discrete bands can increase to over seven without changing thephysical design.

The electrophoresis channels and the optical detection assembly,including the detector and light source, can occupy a volume of about orless than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 2, or 5 ft³.

In some embodiments of the invention, the integrated analytical systemcan include other types of analytical devices. These analytical devicescan include devices for performing mass spectrometry, gaschromatography, liquid chromatography, or any other type of analysisdescribed herein.

The control systems, including the computer and software, can allow fora user to select analysis parameters, control the hardware, and obtaindata. The electronics of the instrument are built in a highly modularfashion using distributed micro controllers to handle peripheral controland management tasks, such as temperature control, switching ofsolenoids, and control of other actuators used in the system. A main“application processor” is responsible for communicating with computerrunning the user interface, and controlling and coordinating theactivities of the system.

The primary user interface can be a 13″ tablet-style, embedded touchscreen mounted in the lid of the enclosure. Night mode with severelyreduced light emission can be an option for the display. Anapplication-specific GUI can overlay the Software instrument control andanalysis software.

The user can turn on an On/Off switch, scan their fingerprint or enterthrough the touch screen. For the Standard User, four buttons can bedisplayed: Insert Cartridge, Load Samples, Start, and Stop. The GUI canbe designed to also provide a gateway to an Expert User mode and anAdmin Mode for replacement of the CAE cartridge, and instrumentcalibration and service. In the Expert Mode, the GUI can also providethe user with various options for data analysis and display and output,USB ports and plug in keyboards can also be enabled per the particularusage scenario and device configuration.

Each peripheral sub system is designed with hardware feedback and safetymechanisms to ensure that the firmware responsible for the operation ofthe module can determine whether the sub-system is operating properlyfrom an electronics point of view. For example, current throughsolenoids and motors is monitored to make it possible to determine thatthe actuator connected and that it is drawing an appropriate amount ofcurrent. (It is possible, for instance, to determine whether a DC motoris stalled, running properly, or disconnected from its load simply bycorrelating the current through the motor to the voltage applied todrive it.)

Similarly, critical fluidics paths are monitored using optical sensorsto determine whether they contain fluid, bubbles or air.

In certain critical systems, such as temperature control, redundantsensors and hardware over temperature protection are integrated toensure that firmware errors cannot cause damage to the system. Further,the temperature sensors used for feedback to the control firmware arebiased to a tightly controlled voltage that makes it possible todetermine whether the sensor is attached or not.

Redundant interlock circuits are employed to ensure operator safety withregards to high voltage, exposure to fluorescence excitation light andother potential hazards.

The system uses CAN bus for communication between the micro controllersin the distributed network. (CAN is a robust serial network.) It enablesfast and reliable communication of messages between modules, and can beset up enable master-slave control schemes that are highly responsive toreal time events in the instrument.

The control of the instrument can be hierarchical. The user interfacecomputer sends high level commands to the main “applications processor”which translates them to sequences of commands for the individual microcontrollers. Each step of such a sequence is executed by sending amessage to the appropriate controller. Commands are always acknowledged,either by the returning the requested data or with a specific responseindicating the receipt of the command. When a module has completed acommand of this latter type, it can send a message to the “applicationsprocessor” indicating whether the completion was successful or not.(There are commands defined that let the “applications processor” queryfor detailed status that may explain any failure.)

Any node on the CAN network can send a message to any other node at anytime. The system uses this to deal with exceptions to normal operations.If for instance, the temperature control module cannot maintain thetemperature within given limits, it can send spontaneous error messageto the “applications processor” to “inform” it of the problem. Similarlythe controller monitoring fluid lines for air or bubbles would send aspontaneous message to enable the appropriate action to be taken by thesystem (or the user) in response to the problem. Depending on thespecifics of the problem, the control application may choose to abortthe run, solicit user action, or simply log and ignore the issue.

When the system is started, all sub-systems run through a basicconfidence test to ensure that all parts of the instrument are operatingproperly. The application can also choose to invoke all or part of thistest at the beginning of each experiment. During operation, each modulemonitors its hardware feedback for signs of errors. If an error isdetected, a message to that effect is sent to the “applicationsprocessor” which can take appropriate action to mitigate the problem(which may cause it to be escalated to the user via the GUI.). Allerrors and other significant events are logged in non-volatile memoryfor later retrieval to assist in troubleshooting and maintenance of theinstrument.

Referring to FIG. 64, the electronic components can include thefollowing: a communications controller (2.1) that communicates with thecomputer and in turn with other electronics modules to relay and monitorexecution of commands; a Solenoid Valve Controller (2.3) that isresponsible for operating the MOVe valves to implement various pumpingand valve control modes. The controller can also operate solenoids forreagent and waste handling, as well as mechanism operation; aTemperature controller (2.3) that runs the thermoelectric devices toimplement thermal cycling and reagent temperature control, as well asresistive heating elements to maintain heated regions (e.g. separationssubsystem); a Motor Controller (2.4) that operates magnet mover andother moving elements; a Sensor Board (2.5) that is an analog anddigital input device that monitors the state of various sensors withinthe system.

Software runs all applications, controls HW and provides GUIfunctionality. Software is designed for the interfacing and control ofinstrumentation and automation components. This architecture is simple,flexible, and designed to allow rapid implementation of laboratoryprotocols. Software utilizes a standardized state machine model thatuses XML files, scripted code, and standard libraries to describe thedevices needed to carry out a given process. A state machine definesoperational logic for a hardware component or software service, definesmethods that are externally available, and provides details aboutcurrent state and data. Initial data analysis is done by MBI softwarewhile the final base calling is done by a third party expert system. Thedata is background subtracted and filtered then spectrally separatedinto its four or five dye channels. Further filtering is performedbefore the data is formed into an AB compatible file and the expertsoftware analysis and call is initiated

Software is composed of four primary sets of code, as follows, referringto Figure FIG. 65:

-   -   A. Core (12.1): A windows service and set of libraries that        implement a set of state machines (one for each hardware        module). A state machine defines operational logic for a        hardware component or software service, defines methods that are        externally available, and provides details about current state        and data.    -   B. GUI (12.2): A simple graphical user interface communicates        with the core to allowing the operator to run and monitor        process scripts.    -   C. Scripts (12.3): Process scripts constitute the operation        logic that controls the hardware to carry out the logic of        defined protocols. Scripts are compiled and run dynamically,        thus providing great flexibility in modifying the logic of the        system.    -   D. Data Analysis (12.4): Initial data analysis is done by MBI        software while the final base calling is done by a third party        expert system. The data is background subtracted and filtered,        then spectrally separated into its four or five dye channels.        Further filtering is performed before the data is formed into an        AB compatible file and the expert software analysis and call is        initiated.

VI. Example of Operation

An example of operating the encased and integrated system is shown inFIG. 86 and FIG. 87.

After powering on the system and logging on by fingerprint recognitionor password, the user can push “Load’ on the touch screen display. Acartridge-handling mechanism can open and the user can insert thecartridge into a mechanism which can then seat and automatically connectall liquid and pneumatic interfaces.

The user can then be prompted to add each sample. Optical barcodes canprovide the chain-of-custody for sample identity and connection toassociated biometric information such as photo, fingerprint, or irisscan. RFID tags may be used to transfer any associated information. Oncethe samples are loaded and the user touches “Go”, the user's interactioncan end until the system reports the results.

The cartridge is then processed by the chassis using reagents on thecartridge. The cartridge can contain all reagents and a positive andnegative control. The processing of the samples in the DNA Profiling cantake about 1¼ hrs. Any biological reaction can be performed in theintegrated and enclosed analysis system. For example, SNP analysis orany other analysis reactions described herein can be performed.

The DNA Profiling cartridge can have four main functional areas held ina frame:

-   -   A. Pre-processing and nucleic acid extraction using a        BeadStorm-based device,    -   B. STR reaction chambers in tubing,    -   C. Microfluidic handling using MOVe micropumps to manipulate        precious reagents and post-process microscale samples and,    -   D. Reagent and waste storage

The chassis Pneumatic subsystem, driven by the Control subsystem,controls all fluidic operations on the cartridge including reagentmetering and distribution, mixing, and bead-based purifications. Thesamples are moved into the ‘BeadStorm-based’ area extracts nucleic acidsfrom large volume raw and/or otherwise unprocessed samples includingswabs and milliliters of liquid using MOVe microvalves to controlpressure-driven flow.

The Thermal subsystem performs the thermal cycling for STR amplificationin the cartridge. The pre-processed samples on beads are moved into theThermal subsystem and the samples eluted off the beads in STR mix, andthermal cycled. The thermal cycler can be based upon the present MBIthermal cycler. The thermal cycled STR products are then diluted intosize standards and pumped by MOVe micropumps to the CAE subsystem.

The CAE subsystem performs the automated separation of the STR productsusing capillary array electrophoresis (CAE) on a reusable CAE cartridgein about 45 min. The CAE subsystem on the chassis can have a highvoltage supply and control, heat controller, and a laser-inducedfluorescent detector with an imaging system capable of high sensitivitydetection and data collection of separated STR products or otherfluorescently labeled biomolecules.

The CAE cartridge performs the actual electrophoretic separation of thelabeled products in an array of 12 separation capillaries. The CAEcartridge has the capillary array, anode and cathode assemblies, windowholder, and a detection window, attached to a frame containing embeddedheating segments. A separation polymer fill device accesses polymer andbuffer on the single use DNA Profiling cartridge and replaces theseparation polymer through an interface at the anode.

When the single-base resolution separation is complete, the digital datais processed to remove background and noise, and perform a spectralseparation by MBI's Trace Analyzer. An expert system based on FSS's IQ3then processes the data to a CODIS metafile. The metafile is queriedagainst local and remote databases and answer if the sample's identifyis produced on the screen in 2 hr after loading.

The MBI integrated single channel ANDE device can have a run time ofthree to three and one half hours depending on the run conditions. Thisincludes the sample entry, lysis, extraction, purification, transfer,elution, thermo-cycling of the 16-plex reaction, transfer, dilution,injection, separation and detection of the products (see FIG. 88A). Thebiggest single segment of time, the thermo-cycling at 100 minutes, canbe significantly reduced. For example, the thermo-cycling time can bereduced to 45 minutes or less. Off system testing has shown good resultswith cycling times of less than one hour. Combining the intelligentprimer design of the PowerPlex with advanced buffer, enzyme and dyechemistries and with efficiencies in the fluidic and separation processcan allow for a processing time of less than two and one half hours (seeFIG. 88B). Processing times less than 1 hr may be achieved (see FIG.88C).

VII. Optical Detection System

The invention provides for a systems approach for the implementation ofan eight channel capillary fluorescence detection and excitationsub-system.

Fluorescently labeled molecules are transported through a sieving matrixin the capillaries (200 μm ID and 50-75 μm OD). Four or more differentdyes are present in the capillaries and the detector part of the systemhas to be able to distinguish their individual spectral signatures.Excitation of all dyes is accomplished using a solid state laseroperating at 488 nm.

As shown in FIG. 89, eight of these capillaries are held in parallel ona glass (or other substrate with similar thermal expansion properties)with epoxy adhesive. The substrate has an aperture that allows theoptical system unrestricted access to the top and bottom of thecapillaries.

The main purpose of the aperture is to limit the amount of material thatis impinged upon by the excitation beam, which minimizes the backgroundfluorescence of the system.

The detection system can be comprised of five elements:

-   -   A. An objective with high numerical aperture (NA=0.65) with        field of view that is large enough to image the eight        capillaries (1.6 mm),    -   B. A laser rejection filter, capable of suppressing the        excitation laser by at least five orders of magnitude.    -   C. A dispersive element (a prism) that separates the images of        the capillaries by wavelength.    -   D. An imaging optic that projects an image of the spectrally        dispersed light from the dispersive element onto the detector.    -   E. An area detector that is capable of capturing images of the        eight parallel, spectrally separated, images of the capilliaries        at a rate of five to ten images per second.

Software that is capable of separating the images of the eightcapillaries and generating the spectral profile of the contents of thecapillary is also an integral part of the system.

An embodiment of the main part of the optical system is show in FIG. 90.The configuration is similar to that of a modern microscope, with an“infinity corrected” design. The objective collects the emitted lightfrom the capillaries and collimates it at its back aperture. A prism isinserted in the collimated path to separate the image of the capillariesinto its spectral components. The imaging optic (known as a “tube lens”in a microscope) then projects that spectrally separated image onto theprimary focal plane of the detector.

As shown in FIG. 91, each capillary forms its own spectrally separatedimage as is shown in the following image. It shows an “end view” of theoptical path projecting each of the capillary images onto the primaryimage plane.

Although the components used in the optical path of the detection systemare chosen to be low noise, there is still an opportunity for thissystem to generate, collect and deliver stray light to the main primaryfocal plane. Much of such light can arrive at that focal plane at anglesthat are substantially different from that of the desired signal.

The alternative optical configuration takes advantage of that byinserting an aperture in the primary focal plane. It then images thatplane to the detector using a relay optic that effectively suppresseslight from outside the desired image of the capillaries. A furtherrefinement on the idea with the aperture, is to place a dichroic mirror,the size of the image, in the primary focal plane. With properly chosencut on wavelength for the coating, this can reject more than 90% oflaser light that may have made it past the laser rejection filter. Themirror is positioned at an angle to direct light toward a detector. FIG.92 shows the dichroic mirror as folding mirror #1.

The purpose of folding mirror #2 is primarily to reduce the foot printof the optical path, and to provide an easy way to align the image onthe detector. The coating on that mirror could, however, also be of thesame kind as that of folding mirror #1, in which case further laserrejection would be possible.

The detector for the system can comprise any photodetector. In oneembodiment, the photodetector is a scientific grade, moderately cooled,two dimensional CCD camera.

The excitation of the fluorescent dyes in the capillaries can be doneusing a 488 nm solid state laser. The laser is projected onto thecapillaries from a direction opposite of the detection system, as shownin FIG. 93. The capillaries are arrayed substantially in a plane. Iflight is directed orthogonally to the plane, the excitation light passesthrough the capillaries and is detected by the optics, interfering withsensitivity of detection. In order to avoid capturing the majority ofthe laser light in the collection system, the excitation is projectedonto the capillaries at an oblique angle with respect to the normalorientation. In this way, the light passing through the capillariesavoids and is not collected by the collection optics.

The optical model shows how the laser beam is scattered in thecapillaries, generating a “plume” of scatter that is outside the captureaperture of the collection optic. (It would be “looking” straight up atthe intersection between the laser beam and the capillaries in the imageabove.)

FIG. 94 shows the excitation path from above. It shows the laser beamslightly diverging from its source. That is because the system useseither an “Engineered Diffuser” or a Powell lens to convert the Gaussianintensity profile of the laser to a uniformly illuminated line of light.This line is then projected (by the cylindrical lenses) directly ontothe capillaries which act as very short focal length cylindrical lenses,effectively focusing the illumination energy inside the themselves.

FIG. 95 shows a simulated image of the capillaries showing theillumination intensity in pseudo color.

Examples Example 1 Operation of a Cartridge for Nucleic AcidPurification

This example refers to the use of a device comprising a cartridge matedto a microchip. The numbers refer to the cartridge of FIG. 3 and FIG. 4mated to a microchip with the circuit architecture of FIG. 5. Thissub-assembly also can be fluidically connected other sub-assemblies inthe instrument of FIG. 6. For reference, a cartridge mated with amicrochip also is shown in FIG. 40 and FIG. 45.

Nucleic acids can be purified from a wide variety of matrices for manypurposes including, but not limited to, genotyping, identification,forensics, gene expression, gene modification, microRNA analysis,ribotyping, diagnostics, or therapeutics. The input sample can be asolid, swab, liquid, slurry, aerosol or a gas.

For molecular diagnostics and forensics, swabs are commonly used. Abuccal swab can be taken using a swab with an ejectable tip and the swabejected into a syringe attached to connection 7 of FIG. 4. Connection 5of FIG. 4 leads by tubing or capillary to a reagent manifold that canselect a single reagent from multiple reagents by opening a full scalevalve or by opening a MOVe valve with the reagents either under pressureor moved by vacuum. MOVe or other micropumps on microchip 2 of FIG. 4can also move the fluids or gases.

In one embodiment, human and other cells in a swab are first lysed usinga buffer with a heated chaotrophic agent and/or other commercial-off-theshelf (COTS) chemistries in a syringe inserted into port 7. The lysateis transported to a DNA isolation chamber (FIG. 4 #3) where paramagneticbeads have been added from a reservoir to adsorb nucleic acids onto thebeads. A moveable magnet is then actuated to capture the beads onto theside of the isolation chamber where they are washed automatically usinga buffer. The purified DNA, still bound to beads, is then pumped througha small diameter tube 250 where multiplexed PCR is performed.Pre-scripted Software automates the complete process. The Softwaredefines a set of communication and command protocols in a standardizedautomation architecture that is simpler, more flexible, and quicker toimplement than other software development approaches. The Softwareimplementation framework is based on core technologies that spanmultiple operating systems, development languages, and communicationprotocols. Software drivers wrap individual smart components of thesystem, greatly reducing the time needed for typical de novo systemsoftware development. This makes it relatively straightforward tointegrate the operation of multiple system modules (pumps, valves,temperature controllers, I/O controllers, etc.) that are either COM- or.NET-based. Software provides a professional quality softwaredevelopment system for prototyping through product release andmaintenance.

While DNA amplification is useful for positive identification ofmicroorganisms, samples can be obtained from a wide variety ofsubstrates and matrices that contain compounds that are inhibitory toDNA amplification reactions. Raw samples are most often complex mixturesthat can include inhibitors such as hemes, metal ions, humic and fulvicacids, chelators, DNases, proteases, and molds. While the initialisolation of target organisms and toxins from the sample matrix by IMSmay remove most of these inhibitors, lysed cell components and lysisagents can also need to be removed or diluted from nucleic acid samplesso that they do not interfere with successful amplification. Raw samplesare otherwise unprocessed samples, and may environmental, biological,and the like.

In one embodiment, a small volume nucleic acid purification is used.These purification methods can be used with a wide range of samples,such as blood, to aerosols, to buccal swabs. Paramagnetic beads can beused in a disclosed device to purify DNA from various sample sources. Inone embodiment a microfluidic microchip can be used to sequence anucleic acid using magnetic beads and reagents to purify nucleic acidproducts for sequencing in microscale reactions. In one embodiment, themicrofluidic microchip is a 24-channel microfluidic microchip.

In one embodiment, polyethylene glycol (PEG)-based nucleic acidpurification is used on carboxylated magnetic beads. ThisPEG-facilitated process can produce yields of over 80% from upstreamimmunomagnetic separations (IMS) captured samples. Development of auniversal sample preparation module (USPM) can partly involve portingthe PEG-based nucleic acid purification onto a device containing acartridge such as the devices shown in FIG. 21 or FIG. 16. In anotherembodiment, Agencourt Orapure or Promega DNA IQ chemistries are used inconjunction with a device of the present invention.

Bead Dispensation and Delivery.

To purify nucleic acids, paramagnetic beads with different surfacechemistries can be mixed in a reagent container. Pressure is thenapplied to send the reagents to connection 5. MOVe microvalves or othervalves may be closed unless referred to as open. To move theparamagnetic beads into the reaction chamber (3), microvalves 180 and150 are opened. The beads are moved through connection 5 into channel 15which leads to junction 190 and microchannel 191. Because microvalves180 and 150 are open and microvalves 200 and 170, and the othermicrovalves, are closed, an open microfluidic connection is frommicrochannel 191 through microvalve 180 to microchannel 181 throughmicrochip 152 to open microvalve 150 and microchip 151 to junction 120.Junction 120 leads to cone 13 and chamber 3, which can be filled withbeads. The volume of beads supplied to chamber 3 can be controlled bytiming the opening of the reagent valves and the microvalves or byfilling and emptying a sample loop connected to the microchip or thecartridge.

Commercial bead based chemistries can be used in the disclosed system,including but not limited to Orapure from Agencourt (Waltham Mass.) andDNA IQ from Promega (Madison, Wis.). Orapure uses a carboxylated beadsurface and SPRI chemistry while DNA IQ is an example of a silica beadand chaotrophic chemistry. Other embodiments of paramagnetic beads orchemistries to process nucleic acids can be used in conjunction with thedisclosed system, including but not limited to beads witholigonucleotides, locked nucleic acids, degenerate bases, syntheticbases, conformation, nucleic acid structures, or other hybridization andspecific capture methods.

Filling Chamber (3) with Beads.

For Orapure or DNA IQ beads, 450 microliters can be moved into chamber(3) using three fills of a 150 microliter sample loop 630 or 631. Amovable magnet 300 attached to actuator 310 can then be moved towardscartridge (1) near the side of 3 to pull the beads to the side ofchamber (3). Magnet size and orientation can be adjusted to generatemagnetic fields appropriate to specific applications. Pressurized aircan then be applied through the reagent manifold with microvalve 180,150, and 110 open. The opening of microvalve 110 connects from junction190 which connects to the reagent manifold through junction 120 andmicrochannels 121 and 101 to connection 100 which leads through channel14 to connection (4) and to waste. The air can move any remaining liquidthrough the circuit. Air or other gases can also be used to dry beads,volatilize solvents, or for bubble-enabled mixing (described herein).

Bubbling of Gas through Chamber (3)

If microvalves 180, 150, and 220 are open, and all other microvalvesclosed, the pressure can force air through chamber (3) to channel 9 anddown channel 19 to junction 210 through microchannels 211 and 221,through open microvalve 220 and microchannel 231 to junction 230,through channel 16 to connection 6 which can be a vent. This sequencecan bubble air or other gases through chamber (3) and can be used to mixreactions in chamber (3) or to change the gas phase.

Moving Liquids and Beads from Chamber (3) to Waste

Liquids and beads can be moved from reaction chamber (3) or any otherlocation to waste. This can be used to wash beads, flush channels, moveliquids or beads to waste. When pressure is applied to connection 6 withmicrovalves 220 and 110 open, and all other microvalves closed, thepressure can force air through channel 16 to junction 230 tomicrochannel 231, through open microvalve 220 and microchannels 222 and221, though junction 210, and channels 19 and 9 into reaction chamber(3) and through junction 120 through microchannel 121, open microvalve110, microchannel 101, channel 14 and to connection 4.

The equivalent effect can be obtained by applying vacuum to connection(4) if connection 6 is a vent without any additional control of airpressure. The air pressure or vacuum can move any liquids in chamber (3)to the waste connection 4. When magnet 300 is close to chamber (3),paramagnetic beads can remain on the side of chamber (3) and the resultis that the liquid is removed. When magnet 300 is far enough fromchamber (3), paramagnetic beads can not remain on the side of chamber(3) and the result is that the liquid and beads are removed.

To clean paramagnetic beads, the beads are pulled to the side of chamber(3) with magnet 300 (see FIG. 6) and the liquid removed to waste. 450microliters of buffer can be dispensed from the reagent manifold andadded to chamber (3) by opening microvalves 180 and 150. The beads canbe released if desired and then recaptured by moving the magnet 300 andthe liquid then removed. This is repeated for a total of three times toproduce beads ready to process samples.

Lysis and Extraction of Nucleic Acids from Cells on the Swab

A swab can be loaded into a syringe barrel inserted into connection 7and then be lysed by addition of lysis buffer through reagent connection5 with microvalves 180 and 170 opened. In some embodiments Orapure orDNA IQ chemistries are used.

Movement of the Lysed Cellular Material to Chamber (3) and Mixing withBeads

The material in the syringe connected to connection 7 can be moved intochamber (3) by applying pressure to the syringe or by applying vacuum tovent 6. When vacuum is used, microvalves 170, 150, and 220 are opened.The vacuum connects through microchannels 231, 221, 211, and channels 9and 19 through chamber (3), microchannels 151, 152, 171, and 161 to pullmaterial from connection 7 into chamber (3). When paramagnetic beads areloaded and cleaned in chamber (3), the lysed sample material mixes withthe beads in chamber (3) with the magnet is the far position.

Purification of Nucleic Acids on the Beads

The paramagnetic beads are then incubated with the lysed sample.Continued air or gas flow can aid mixing. The magnet 300 is then movedto the closed position and the beads are captured on the wall of chamber(3). The sample lysate can then be removed from chamber (3) to waste andmultiple volumes of wash solution added according to manufacturers'specifications for the Orapure chemistry or DNA IQ chemistry. The samplecomponents on the beads have now been purified and are ready forreactions in the cartridge or exporting to the sample productconnection. In one embodiment the beads are used to enrich a nucleicacid component from a sample.

Exporting Samples through the Sample Product Connection 8

The purified sample components on the beads can be moved to connection 8by applying pressures on reagent connection 5 with microvalves 180, 150,and 130 open. In one embodiment, connection 8 is connected with reactionchannel 250 such as C-flex tubing (Cole Parmer) and additional reactionsare performed in the reaction channel.

Multiplexed PCR Amplification of STR Markers

DNA amplification can be performed by PCR amplification. The presentinvention enables PCR reactions as well as many other DNA amplificationand modification reactions. The reactions can be performed in chamber(3), in reaction channel 250 attached to connection 8 which can be atube 250 (FIG. 3, FIG. 4, FIG. 6), or in another device or microdeviceconnected to tube 250. This demonstrates the utility of the samplepreparation for DNA reactions including thermal cycling.

Capture of Nucleic Acid Containing Beads in a Reaction Channel

The purified DNA output through the sample product connection 8 is movedinto a reaction channel 250 at end 251 by applied pressure oralternatively through vacuum applied to end 252. An actuator 330 moves amagnet 320 under software control into a position close to bead captureregion 340. Fixed magnets of different sizes and shapes (such as rareearth magnets) as well as electromagnets or superconducting magnets canbe used. As the solution containing the beads moves through region 340,the magnetic field attracts the beads to the side of the reactionchannel and holds them in place. The fluid is then followed by airpressure through reagent connection 5 leaving the beads region 340 inair.

Addition of Reagents and Movement of Samples into Reaction Region

Reagents can be added from the reagent manifold as described. In oneembodiment, reagents are added from end 252 of reaction channel 250. End252 is attached to a microfluidic microchip 500 comprising microvalves510, 520, 530, and 540. Any three microvalves such as 510, 520, and 530or 510, 520, and 540 can form a pump. Microvalve 530 connects through amicrochannel to a downstream device 535, which can connect to tubingleading to a reagent reservoir. Microvalve 540 connects through amicrochannel to downstream device 545, which can connect to tubing thatleads to a reagent reservoir.

Reaction mixes (such as at least one DNA polymerase, dNTPs, buffer and asalt) including but not limited to master mixes and primers, (such asassay-specific primers or broadly applicable primer sets for multipletarget pathogens), or complete PCR master mixes such as PowerPlex 16from Promega (Madison, Wis.) or IdentiFiler or MiniFiler from AppliedBiosystems (Foster City, Calif.) in reagent reservoir 600 can bedelivered by a micropump formed by microvalves 530, 520, and 510 throughtubing 610 and microchannels 531, 521, 511, and 512, into end 252 ofreaction channel 250, as shown in FIG. 6. MOVe microvalves can preciselyposition fluids and move the fluid to region 340 where the reaction mixencounters the beads comprising nucleic acids. Magnet 320 is moved awayfrom reaction channel 250 by actuator 330 which releases the beads fromthe inner surface of the reaction channel 250. The MOVe microvalves onmicrochip 500 pump the beads into device 400 with an area of reactionchannel 250 forming temperature controlled region 350. The region 350can be held at isothermal temperatures or thermal cycled or other variedas is well known to one skilled in the art. The region 350 can be atemperature modulator or thermally coupled to a temperature modulator.

FIG. 7 shows a temperature control device 400 that is capable of thermalmodulation using a temperature modulator for heating and cooling tothermocycle the reaction channel. In one embodiment the temperaturemodulator comprises a Peltier module, infra-red module, microwavemodule, a hot air module or a light module. In another embodiment a PCRreaction sample is moved inside the reaction channel past one or moreconstant temperature zones.

FIG. 9 shows the amplification of PowerPlex 16 STR reactions that havebeen prepared in a cartridge (1) from buccal swab samples and processedin reaction channel 250 using the temperature control device 400 in FIG.7. The STR markers are amplified from standard conditions with Mgoptimized for the apparatus 1000.

The temperature control device 400 can also have a detector 410. Thedetector can detect optical detection such as absorbance, fluorescence,chemiluminescence, imaging, and other modalities well known to oneskilled in the art or measurement such as IR, NMR, or Ramanspectroscopy. The detector can comprise a light source is used to excitea fluorescent or luminescent dye in the PCR reaction sample, and theexcitation light is sensed with a photodetector (such as a CCD, CMOS,PMT, or other optical detector). In one embodiment the light source is acoherent light source, such as a laser or a laser diode. In anotherembodiment the light source is not a coherent light source, such as alight emitting diode (LED) or a halogen light source or mercury lamp.

For nucleic acid amplification, real-time PCR is one example of anucleic acid assay method that can be performed in tube 250 intemperature controlled region 350 and detected with detector 410.

Example 2 Universal Sample Preparation

The previous example illustrated one embodiment in which the disclosedapparatus can be used to prepare samples for analysis and showed oneexample of STR amplification. Another embodiment involves the use of aUniversal Sample Preparation Module (USPM). The USPM device can consistof a sample processing cartridge (1), accompanying apparatus to operatethe cartridge, a microprocessor, and software that can readily beinterfaced to downstream analytical devices. In one embodiment the USPMcan be tightly integrated with analytical devices to form a modularsample-to-answer system. The cartridge can be configured as a disposablesingle-use device that can process swabs or liquids (including aerosolsamples) for field monitoring processes, or as a reusable, flow-throughformat for remote operations with rare positives. Target specificity ofthe USPM is imparted through the use of specific antibodies (that bindselected targets) attached to paramagnetic beads; different cartridgescan be supplied with various mixtures of targets.

A USPM can use a multistep fully automated process to prepare biologicalsamples for downstream analysis. One example in FIG. 18 can use swabs orliquids; the operator can select the sample type and then insert samplesinto input port(s). The first step can apply immunomagnetic separations(IMS) to capture, concentrate, and purify target molecules from solutiononto paramagnetic beads. Targets already tested include cells, spores,viruses, proteins, or toxins. For toxin and protein detection, or foruse as a triggering device, the captured targets from the IMS can beexported directly to the downstream analytical device. For nucleic aciddetection, the second step can lyse the cells or spores to release theDNA and/or RNA using mechanical or other lysis techniques. The thirdstep, nucleic acid purification, can adsorb, concentrate, and purify thenucleic acids onto a second set of paramagnetic beads and output thebeads with nucleic acid, or purified desorbed nucleic acid, fordownstream analysis.

Referring to cartridge (1), the immunomagnetic separation can beperformed by using reagent beads that have antibodies or otherimmunomagnetic, affinity magnetic, or surface chemistry magneticseparations. For example, immunomagnetic beads with antibodies can beadded to cartridge (1) to capture, purify, and concentrate cells,viruses, spores, toxins and other biomolecules onto bead.

Upstream sample processing for the USPM can be done in the samplepreparation devices, which can process samples over 0.6 mL in amicrofluidic cartridge (1) (FIG. 21). The sample processing cartridge,about 1 in cubed dimension, (FIG. 3, FIG. 21) was developed toautomatically remove collected buccal cells from a swab, lyses thecells, and purifies released cellular DNA on magnetic beads. The beadbeds are typically 100 nL and can be used for downstream STR analysiswith microfluidics devices or full scale qPCR reactions.

The sample preparation device uses a MOVe microvalve microchipinterfaced with the bottom of the cube (FIG. 3, arrow labeled 2) todirect pressure-driven flows consisting of fluids, beads, and samplesamong the reagent and reaction reservoirs. The MOVe microvalves replaceconventional valves and tubing between the reservoirs, thereby providinga non-leakable, directable fluid transport and enable miniaturization ofthe entire cube and sample preparation device.

This sample preparation device technology has been used to automate DNAextraction from buccal swabs as described above. FIG. 10 shows automatedpreparation of DNA from 25 uL of blood in the automated samplepreparation device using pressure driven flows, vibrational mixing, MOVevalves, actuated magnets, and magnetic beads. The fully automatedprocess produced DNA ready for STR analysis in less than five minutes.

An automated system has been developed for capturing, concentrating, andpurifying cells, viruses, and toxins from liquid samples (1-10 mL) usingmagnetic beads coated with antibodies specific to targets of interest.Thus, a variety of targets have been concentrated and purified with thisautomated system. Using this approach, E. coli cells were captured anddetected at cell concentrations as low as 15 cells/mL/sample (FIG. 27).Similar results of greater than 90% capture efficiency were obtainedusing Bacillus spores, Gm⁺ and Gm⁻ vegetative cells, a model virus(bacteriophage fd), SEB, and ovalbumin as targets. Purified samples canbe further processed in the sample preparation device (e.g., lysis andnucleic acid purification), moved onto a microchip for analysis, or usedwith an off-chip PCR/qPCR device.

IMS capture has been shown to work well in complex samples such asaerosols and in the presence of biological clutter (See U.S. PatentPublication No. 20080014576, herein incorporated by reference in itsentirety). For clutter, we showed that up to 10⁵-fold levels of addedbacteria produced only a two-fold reduction in capture efficiency. Forcomplex samples, add-back experiments using many different aerosolsamples established that aerosol samples reduce the binding of B. cereusspores to IMS beads by less than 50%. Therefore, there is less than atwo-fold loss of sensitivity in complex, real-world samples.

IMS has been used to capture, concentrate, and detect toxins. We havedeveloped IMS assays for ovalbumin and SEB, multiplexed the assays, anddeveloped two generations of completely integrated microfluidic systemsthat automate the IMS assays. Less than 10 ng of SEB can be reliablydetected in a one mL samples with no interference from closely relatedStaphylococcal enterotoxins.

IMS can:

-   -   A. Select target organisms from samples with high backgrounds of        interferents (selectivity),    -   B. Discriminate between two different strains or species of        bacteria (specificity),    -   C. Effectively capture cells and toxins across a wide range of        concentrations from a wide range of samples (sensitivity,        robustness)    -   D. Reduce target sample volume significantly, from mL to nL        volume

The instant invention and the apparatus and methods are capable ofimplementing IMS and coupling it to nucleic acid extractions.

The next step in the USPM is the lysis of the captured target when it isa cell, virus, prion, or spore. Lysis of spores is particularlychallenging. A MagMill or magnetically driven lysis or homogenizingdevice has been developed for efficient lysis of Bacillus and otherspores, as well as vegetative cells. The MagMill consists of a rapidlyrotating magnet 2000 actuated by a motor 2001 (FIG. 60) that drivesrotation of another magnet 2002 contained within a sample-containingvessel 2003 or compartment (FIG. 61). The magnet 2002 contained withinthe sample-containing vessel can have any shape. For example, the magnetcan have a bar, spherical, cylindrical, rectangular, oval, hexagonal, orpropeller shape. Alternatively, the magnet can have holes through it,such that liquid may be forced through the holes and increase the shearforce applied to the sample when the magnet is rotated by a magneticfield. The same basic components can be miniaturized, incorporated intoa microfluidic format, or connected to a microfluidic format. Theoverall effect is analogous to a magnetic stir plate, with the samplebeing rapidly vortexed within the sample tube. Using magnetically drivensample agitation by MagMill treatment, spore lysis is achieved withoutsilica, zirconia or other beads. Lysis may be accomplished by shearforces generated as the spore passes between the magnet and the vesselwalls. The magnet can rotate at a rate of greater than about 10, 50,100, 150, 200, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, or 10000rpm.

This device disrupts spores with similar efficiency as traditional beadbeating that employs silica/zirconia beads (FIG. 62). Spores (3.2×10⁷)were lysed in a volume of 1 ml with viability was determined by platingon Tryptic Soy Agar; results are an average of two separate experimentseach run with duplicate samples (total n=4). The non-viability ofmagnetically-driven spore lysates was 93% compared to traditional beadbeating (BioSpec beater) lysates using either Zirconia/silica which was96% or silica beads which was 80%. The same pattern was confirmed byqPCR

The advantage of using the MagMill (versus traditional bead beating) isthat the design is more mechanically robust and thus able to withstandmany cycles of use without failure, and samples can be lysed using justthe agitation of the magnet in the sample, without the need forinclusion of silica/zirconia beads that have been shown to bind releasedDNA causing a loss in follow-on detection sensitivity. The basicfeatures of the MagMill can be reconfigured in a miniaturized formatthat can be integrated into a sample preparation device. The system canpotentially be down-sized to fit into a microfluidic microchip. Despitechanges in configuration, however, the principle driving lysis, that ofa rapidly rotating magnet contained within a sample vessel, remains thesame.

Example 3 Coupling of a Sample Preparation Device with a Microchip-BasedSample Cleanup and Separation

FIG. 34 and FIG. 35 show a device with a cartridge (2907) and microchip(2901) that was designed to incorporate the Forked Injector design, asshown in FIG. 32, a gel filling manifold (2903), and associatedcomponents. The cartridge is fluidically connected to a pneumaticsmanifold and tubing (2905). Different configurations of the injectordesign, separation channel length and separation polymer were tested.FIG. 36 show an electropherogram of an M13 T track injected andseparated on a microchip channel using the Forked Cathode injector, withsample detection on a confocal microscope breadboard system. The samplewas injected uniformly with short and long DNA fragments representedequally. The results show that an M13 T track DNA ladder can beuniformly injected and single base pair resolution can be obtained outto approximately 330 base pairs in less than 20 minutes. Higher samplesignal strengths were obtained compared to injections using aconventional twin T design. When integrated with a detection system, themicrochip is held at a constant 50° C. in order to obtain separationswith good resolution.

Using these processes, excellent results were obtained for MOVeintegrated, field amplified stacking injections of liquid samples (FIG.37). This data was generated with all sample loading, manipulation andinjection processes carried out under software control using MOVemicrovalves. The data has been minimally processed, color corrected froma detector that uses eight diode channels to four dye traces.

One embodiment of a microchip that combines the forked cathode with aMOVe sample preparation device is shown in FIG. 38. This devicecomprised additional processes that enable integration with the rest ofthe system, i.e., the sample preparation device (1000 shown in FIG. 22),the reaction channel (250 shown in FIG. 6), and the output of the STRpurification as described in the STR example. FIG. 38 shows a forkedcathode with MOVe fluidics and shuttle sample loading for integrationwith post amplification STR purification system. The parts are:1—Reagent input port, 2—Reagent pump head, 3—Sample input port, 4—SizeStandard/eluent input port, 5—Capture valve, 6—Waste port, 7—Elutionvalve, 8—Sample waste port, 9—Cathode, 10—Cathode port, 11—Sample valve,12—Sample port, and 13—Separation channel. The anode port, which isdownstream of the channel, is not shown.

The sample to be separated is introduced as a bead solution in ethanol.This can be the purified reaction products on beads output as describedabove. In one embodiment, the sample is an STR reaction. In otherembodiments, the sample can be nucleic acid fragments of differentlengths produced by other reaction chemistries including DNA sequencingby Sanger chemistry. The solution containing the sample is flowed fromthe Sample input port to the Sample waste port with the Capture valveand other intervening valves open. The open Capture valve facilitates aslowing of the stream flow and bead capture by a fixed magnet placedabove or below the valve. The ethanol solution is completely run throughthe system followed by air yielding a relatively dry and clean bead bed,with purified products, in the valve. At this point the valve is closedand reopened (in coordination with other valves) to fill it eluentsolution from the associated port. For an STR analysis or other analyzeswhere an internal size standard is needed, the eluent can contain a sizestandard. The solution is moved between the Elution valve and theCapture valve to facilitate mixing, ending with the solution in theElution valve. The Sample valve is then opened in coordination with theElution valve closing to “shuttle” the sample through the sample channelleaving it filled. The sample FASS injection is carried out aspreviously described. An additional noteworthy function of the device isthat in one embodiment the Reagent input port and Reagent pump are usedto provide metered STR reaction premix to the reaction channel (250shown in FIG. 6) after the swab extraction of DNA on the samplepreparation device; in other embodiments, the device can provide othernucleic acid reaction reagents such as cycle sequencing mixture orprovide PCR reagents to perform a PCR amplification followed byproviding cycle sequencing reagents to perform cycle sequencing withbead-based cleanup reactions integrated as needed. Other chemistrieswill be apparent to one skilled in the art.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. For example, any MOVevalve, pump, router, or other MOVe device described herein can bereplaced with any pneumatically actuated valve, pump router or otherdevice. It should be understood that various alternatives to theembodiments of the invention described herein can be employed inpracticing the invention. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1. A system that fits within an enclosure of no more than 10 ft.³comprising: (a) a sample preparation module adapted to capture ananalyte from a non-microfluidic volume on a capture particle and routethe captured analyte through a microfluidic channel; (b) a reactionmodule comprising a reaction chamber in fluidic communication with themicrofluidic channel adapted to immobilized the captured analyte andperform a biochemical reaction on the analyte in a non-microfluidicvolume to produce a reaction product; and (c) an analysis module influidic communication with the reaction chamber adapted to perform ananalysis on the reaction product.
 2. The system of claim 1 configured tocapture the analyte, perform a biochemical reaction on the analyte, andperform an analysis on the product in less than 4 hours.
 3. The systemof claim 1 further comprising a data analysis module configured toreceive data about the analysis from the analysis module and comprisingexecutable code that transforms the data and outputs a result of theanalysis.
 4. The system of claim 1 further comprising a processingmodule in fluidic communication with the reaction chamber and theanalysis module and adapted (1) to route the reaction product through asecond microfluidic channel into a non-microfluidic processing chamber;(2) process the reaction product and (3) route the processed reactionproduct into the analysis module.
 5. The system of claim 1 wherein thesystem fits within an enclosure of no more than 8 ft³.
 6. The system ofclaim 1 wherein the system fits within an enclosure of no more than 5ft³.
 7. The system of claim 1 wherein the system fits within anenclosure of no more than 2½ ft³.
 8. The system of claim 1 wherein thesample preparation module is adapted to release the analyte from a cell.9. The system of claim 1 wherein the capture particle is a magneticallyresponsive capture particle and the reaction module comprises a sourceof magnetic force configured to immobilized the captured analyte. 10.The system of claim 1 wherein the reaction module is adapted to performthermal cycling.
 11. The system of claim 1, wherein the system is aclosed system.
 12. The system of claim 1, wherein the system is batteryoperated.
 13. A system comprising a cartridge cover, a cartridge and apneumatic manifold wherein the cartridge can be releaseably engaged withthe cartridge cover and the pneumatic manifold, wherein the cartridgecomprises one or more pneumatically actuated valves and one or moremicrofluidic channels, wherein the pneumatic manifold and the cartridgecover are each fluidically connected to at least one pressure source,and wherein the pneumatic manifold and the cartridge cover are eachadapted to control fluid flow within the cartridge.
 14. The system ofclaim 13, wherein the pneumatic manifold is adapted to actuate thepneumatically actuated valves and the cartridge cover is adapted toapply pressure to one or more chambers in the cartridge.
 15. A systemcomprising: a) a disposable cartridge comprising at least one set offluidic chambers including a sample chamber, a mixing chamber and athermal cycling chamber in fluid communication with each other, and areagent card comprising reagents for performing a chemical reactioninvolving thermal cycling, wherein the reagent card is configured to becarried on the cartridge in a closed configuration and to be moved intofluid communication with the at least one set of fluidic chambers; b) anactuator assembly configured to move fluids between chambers when thecartridge is engaged with the actuator assembly; c) a thermal cyclerconfigured to cycle temperature in the thermal cycling chamber when thecartridge is engaged with the actuator assembly; d) a capillaryelectrophoresis assembly configured to accept a sample from cartridgewhen the cartridge is engaged with the actuator assembly and to performcapillary electrophoresis on the sample; and (e) a computerized controlsystem configured to control the actuator assembly, the thermal cyclerand the capillary electrophoresis assembly.
 16. A cartridge comprising:a) a fluidic manifold comprising a fluidic side and a reagent card sidewherein the fluidic manifold comprises: (i) at least one set of fluidicchambers, each chamber comprising a port on the fluidic side; (ii) atleast one of thermal cycling chamber comprising at least one port; (iii)at least one of exit port; (iv) a slot on the reagent card side adaptedto engage a reagent card, wherein the slot comprises a plurality of slotchannels comprising cannulae on the reagent card side and communicatingbetween the two sides; b) at least one microfluidic chip comprising: (i)at least one fluidic circuit; (ii) a plurality of ports in fluidcommunication with the fluidic circuit; (iii) at least one pneumaticallyactivated diaphragm valve configured to regulate fluid flow within thefluidic circuit; wherein the at least one chip is engaged with thefluidic manifold so that the ports in the at least one chip are in fluidcommunication with the ports of the chambers and the slot channelswherein each fluidic chamber is in fluid communication with at least oneother fluidic chamber and each cannula is in communication with afluidic chamber; and c) a reagent card engaged with the slot, whereinthe card comprises a plurality of reagent chambers comprising reagents,each aligned with at least one of the cannulae and adapted to take afirst engagement position wherein the reagent chambers are not puncturedby the cannulae and a second engagement position wherein the reagentchambers are punctured by the cannulae, thereby putting the reagentchambers in fluid communication with the fluidic circuit.
 17. Thecartridge of claim 16 wherein the reagents comprise reagents forperforming PCR.
 18. The cartridge of claim 17 wherein the reagentscomprise primers for amplifying a plurality of short tandem repeats. 19.The cartridge of claim 16 wherein the at least one set of fluidicchambers is a plurality of sets of fluidic chambers.
 20. The cartridgeof claim 16 wherein the fluidics manifold further comprises at least oneauxiliary fluidic channel on the fluidic side of the manifold, the atleast one chip is a plurality of chips and fluidic circuits in each ofthe plurality of chips are in fluidic communication with fluidiccircuits of at least one other chip through the auxiliary fluidicchannel.
 21. The cartridge of claim 20 wherein further comprising agasket between the chips and the manifold, wherein the gasket seals thechannels on the manifold.
 22. The cartridge of claim 16 wherein thefluidic chambers comprise a distribution chamber, a capture chamber, asample chamber, and a clean-up chamber.
 23. The cartridge of claim 16wherein at least one fluidic chamber comprises magnetically responsiveparticles.
 24. The cartridge of claim 16 wherein the at least one set offluidic chambers is at least 4 sets or at least 8 sets.
 25. Thecartridge of claim 16 wherein the chips comprise at least one diaphragmvalve.
 26. A system comprising: a) a pneumatic assembly comprising: (i)a pneumatic manifold adapted to removably engage the cartridge of claim16 on the fluidic side, wherein the pneumatic manifold comprises aplurality of pneumatic ports configured to engage pneumatic channels inthe at least one microfluidic chip and activate the diaphragm valves;and (ii) a pressure source configured to supply positive or negativepressure to the pneumatic channels; b) a cartridge activation assemblyadapted to engage the cartridge on the reagent card side; wherein thecartridge activation assembly comprises: (i) a reagent pneumaticmanifold comprising a pneumatic side and reagent card side, wherein thereagent pneumatic manifold comprises reagent pneumatic manifold channelscommunicating between the two sides and comprising a cannula on thereagent card side; (ii) a pressure source configured to supply positiveor negative pressure to the reagent pneumatic manifold channels; and(iii) a clamp configured to move the reagent card from the firstengagement position to the second engagement position, wherein clampingresults in the cannulae of the reagent pneumatic manifold puncturing thereagent chambers and putting the reagent chambers in communication withthe pressure source; c) a thermal cycler configured to cycle temperaturein the at least one thermal cycling chamber when the cartridge isclamped; d) a capillary electrophoresis assembly comprising: (i) atleast one separation channel fluidically engaged with the exit port whenthe cartridge is clamped; and (ii) an optical sub-assembly configured todetect signal from the at least one separation channel; and (e) acomputerized control system configured to control pneumatic assembly,the cartridge activation assembly, the thermal cycler and the capillaryelectrophoresis assembly.
 27. The system of claim 26 wherein clampingseals the chamber ports and the slot channels with the at least onemicrofluidic chip.
 28. The system of claim 26 wherein the cartridgeactivation assembly further comprises at least one heater configured toheat at least one of the fluidic chambers when the reagent pneumaticmanifold is engaged with the cartridge.
 29. The system of claim 26wherein the cartridge activation assembly further comprises movablemagnets configured to move into and out of a position wherein themagnets exert a magnetic force on at least one fluidic chamber.
 30. Thesystem of claim 26 wherein the cartridge activation assembly furthercomprises sensors configured to detect the presence of a sample in asample chamber of the fluidic manifold.
 31. The system of claim 26wherein the thermal cycler comprises a Peltier device.
 32. The system ofclaim 26 comprised in a portable case.
 33. The system of claim 32wherein the case has an internal volume of no more than 10 ft³.
 34. Thesystem of claim 32 wherein the case has an internal volume of no morethan 8 ft³.
 35. The system of claim 32 wherein the case has an internalvolume of no more than 2½ ft³.
 36. An article in computer readable formcomprising code for operating the system of claim
 26. 37. A methodcomprising: producing, from a sample comprising at lest one cellcomprising DNA, a computer file identifying a plurality of STR markersin the DNA, wherein the method is performed in less than 4 hours. 38.The method of claim 37 wherein the method is performed in less than 3hours.
 39. The method of claim 37 wherein the method is performed inless than 2 hours.
 40. The method of claim 37 wherein producingcomprises extracting the DNA from the at least one cell, amplifying theSTR markers from the DNA, performing capillary electrophoresis on theamplified markers, detecting the amplified markers, and performingcomputer analysis on the detected amplified markers to identify themarkers.
 41. The method of claim 37 wherein the plurality of STR markersis at least 5 STR markers.
 42. The method of claim 37 wherein theplurality of markers are CODIS STR markers.
 43. The method of claim 42wherein the plurality of STR markers is at least 5, 10, or 13 CODIS STRmarkers.
 44. The method of claim 37 wherein the at least one cell is aplurality of cells.
 45. The method of claim 37 wherein the sample is aforensic sample.
 46. The method of claim 37 performed at the site of asample collection.
 47. The method of claim 37 wherein the samplecomprises blood.
 48. The method of claim 37 wherein the sample comprisesa cheek swab.
 49. The method of claim 37, wherein the method is carriedout by a system as described in any of claim 1-15 or 26-35.
 50. A systemconfigured to perform a method, wherein the method comprises: producing,from a sample comprising at least one cell comprising DNA, a computerfile identifying a plurality of STR markers in the DNA, wherein themethod is performed in less than 4 hours.
 51. A method comprising: a)providing a system comprising: i) a disposable cartridge comprising atleast one set of fluidic chambers including a sample chamber, a mixingchamber and a thermal cycling chamber in fluid communication with eachother, and a reagent card comprising reagents for performing a chemicalreaction involving thermal cycling, wherein the reagent card isconfigured to be carried on the cartridge in a closed configuration andto be moved into fluid communication with the at least one set offluidic chambers; ii) an actuator assembly configured to move fluidsbetween chambers when the cartridge is engaged with the actuatorassembly; iii) a thermal cycler configured to cycle temperature in thethermal cycling chamber when the cartridge is engaged with the actuatorassembly; iv) a capillary electrophoresis assembly configured to accepta sample from cartridge when the cartridge is engaged with the actuatorassembly and to perform capillary electrophoresis on the sample; and v)a computerized control system configured to control the actuatorassembly, the thermal cycler and the capillary electrophoresis assembly;b) moving of the reagent card into fluid communication with at least oneset of fluidic chambers; c) providing a sample comprising a nucleic acidmolecule to a sample chamber; and d) operating the system to amplify anddetect at least one nucleic acid sequence in the sample.
 52. The methodof claim 51 comprising providing each of a plurality of samples to adifferent sample chamber.
 53. The method of claim 51 wherein the time ittakes to go from step b) to step d) is less than 4 hours.
 54. The methodof claim 51 comprising amplifying and detecting a plurality of nucleicacid sequences in the sample.
 55. The method of claim 54 wherein theplurality of nucleic acid sequences comprise short tandem repeats. 56.The method of claim 56 wherein the short tandem repeats comprise aplurality of Combined DNA Index System (CODIS) markers.
 57. The methodof claim 56 wherein the CODIS markers comprise a plurality of markersselected from AMEL, D3S1358, THO1, D21s11, D18s51, D5s818, D13s317,D7s820, D16s539, CSF1PO, vWA, D8S1179, TPOX and FGA.
 58. The method ofclaim 51 wherein the system is a system of claim
 26. 59. The method ofclaim 51 wherein the sample is a forensic sample.
 60. The method ofclaim 51 wherein the sample is selected from a buccal swab, blood, hairor semen.
 61. The method of claim 51 wherein the sample is a raw sample.62. The method of claim 51 further comprising transporting the system toa forensic site.
 63. An optical system comprising: a) a plurality ofoptically transparent channels; b) a light source configured to directto the plurality of optically transparent channels; c) a dispersiveelement that disperses light passing through the optically transparentchannels in a wavelength dependent manner; and d) a detector configuredto receive the dispersed light.
 64. The system of claim 63, wherein theplurality of optically transparent channels comprises at least eightcapillaries.
 65. The system of claim 63, wherein the opticallytransparent channels are aligned in a first plane and the dispersiveelement disperses light along a second plane, wherein the first planeand the second plane are different.
 66. The system of claim 65, whereinthe first plane is orthogonal to the second plane.
 67. An optical systemcomprising: a) an excitation source configured to direct excitationlight to an object; b) a carrier for an object, wherein the object emitslight other than excitation light when excited by the excitation energy;c) a rejection filter configured to filter out excitation energy and toallow transmission of the emitted light; d) an imaging lens configuredto focus the emitted light; e) a dichroic mirror substantiallytransparent to the excitation energy and configured to reflect emittedlight to a detector; f) a focusing system comprising at least one lensconfigured to focus light reflected from the dichroic mirror; and g) aphotodetector (CCD camera) configured to receive the reflected light.68. The optical system of claim 63 wherein the excitation lightcomprises light of a wavelength between 0.3 microns and 1 micron. 69.The optical system of claim 67 wherein the carrier comprises an array ofcapillary tubes and the object comprises a fluorescent species.
 70. Theoptical system of claim 67wherein the mirror reflects emitted light andan angle between about 5 degrees and about 10 degrees off an incidentangle.
 71. The optical system of claim 67wherein the dichroic mirrorfurther comprises a portion that transmits substantially all light. 72.The optical system of claim 67wherein the focusing system comprises atleast one folding mirror.
 73. The optical system of claim 67wherein thephotodetector comprises a CCD camera.
 74. The optical system of claim 67further comprising a prism located between the object and the imaginglens.
 75. An optical system comprising: a) an array of capillary tubesaligned substantially parallel and substantially in a plane; b) anexcitation assembly comprising an excitation source and configured todeliver excitation light from the excitation source to the array,wherein the light delivery assembly is configured (i) to deliver a thinband of light that covers the array and (ii) to deliver the light to thearray at an angle other than 90 degrees to the plane; c) a collectionlens configured to collect light emitted from the array by objects inthe array excited by the excitation light; wherein the excitationassembly and the collection lens are configured with respect to thearray so that excitation light passing through the array substantiallyavoids collection by the collection lens.
 76. The optical system ofclaim 75 wherein the angle is between about 10 degrees and about 85degrees.
 77. An instrument comprising: a) a microfluidic componentcomprising a plurality of intersecting microfluidic channels and atleast one controllable valve configured to regulate flow of fluidbetween the intersecting channels; and b) a non-microfluidic componentcomprising a plurality of non-microfluidic chambers, wherein eachnon-microfluidic chamber is fluidically connected to at least one of themicrofluidic channels; wherein the instrument is configured to flowfluid from at least one non-microfluidic chamber into anothernon-microfluidic chamber through a microfluidic channel and flow isregulated by at least one valve.
 78. The instrument of claim 77 whereinthe plurality of non-microfluidic chambers comprises at least threechambers and the at least one valve selectively directs fluid from onechamber to either of the at least two other chambers.
 79. The instrumentof claim 77 further comprising a pump to pump fluid from anon-microfluidic chamber into a microfluidic channel.
 80. The instrumentof claim 77 wherein at least one valve is a diaphragm valve.
 81. Theinstrument of claim 80 wherein the pump is a diaphragm pump comprising aseries of three diaphragm valves.
 82. The instrument of claim 77 whereinthe microfluidic component comprises a monolithic device.
 83. Theinstrument of claim 77 wherein the combination of the microfluidiccomponent and the non-microfluidic component define a fluidic circuitand the instrument comprises a plurality of fluidic circuits.
 84. Theinstrument of claim 77 wherein the non-microfluidic component furthercomprises a particulate capture agent.
 85. The instrument of claim 84wherein the particles are responsive to a magnetic field and theinstrument further comprises a magnet configured to immobilize theparticles.
 86. A device comprising a plurality of non-microfluidicchambers fluidically connected to a common microfluidic channel.
 87. Amethod comprising: a) providing a device comprising: i) a microfluidiccomponent comprising a plurality of intersecting microfluidic channelsand at least one controllable valve configured to regulate flow of fluidbetween the intersecting channels; and ii) a non-microfluidic componentcomprising a plurality of non-microfluidic chambers, wherein eachnon-microfluidic chamber is fluidically connected to at least one of themicrofluidic channels; wherein the instrument is configured to flowfluid from at least one non-microfluidic chamber into anothernon-microfluidic chamber through a microfluidic channel and flow isregulated by at least one valve; and wherein a first non-microfluidicchamber comprises a first volume of sample comprising an analyte; b)providing an amount of particulate capture agent in the firstnon-microfluidic chamber to bind a selected amount of analyte from thesample; c) moving the particulate capture agent bound to the analytethrough a microfluidic channel in the microfluidic device to a secondnon-microfluidic chamber; d) contacting the particulate capture agentbound to the analyte with a reagent in a second non-microfluidicchamber; and e) performing a chemical reaction on the analyte using thereagent.
 88. The method of claim 87 wherein contacting comprises flowingthe reagent from a third non-microfluidic chamber through a microfluidicchannel in the microfluidic device into the second non-microfluidicchamber.
 89. The method of claim 87 wherein the particles are responseto magnetic force and the method further comprises immobilizing theparticulate capture agent bound to the analyte in the instrument with amagnetic force.
 90. The method of claim 87 comprising suspending theparticulate capture agent bound to the analyte in the instrument in avolume at least an order of magnitude smaller than the sample volume.91. A method comprising: a) performing a first chemical reaction on ananalyte in a first chamber which is a non-microfluidic chamber toproduce a first reaction product; and b)moving the first reactionproduct through a microfluidic channel into a second chamber which is anon-microfluidic chamber and performing a second chemical reaction onthe first product to create a second reaction product.
 92. A methodcomprising: a) performing a first chemical reaction on an analyte in afirst chamber which is a non-microfluidic chamber to produce a firstreaction product; and b) moving the first reaction product through amicrofluidic channel into a second chamber which is a microfluidicchamber and performing a second chemical reaction on the first productto create a second reaction product.
 93. A method comprising: a)performing a first chemical reaction on an analyte in a first chamberwhich is a microfluidic chamber to produce a first reaction product; andb) moving the first reaction product through a microfluidic channel intoa second chamber which is a non-microfluidic chamber and performing asecond chemical reaction on the first product to create a secondreaction product.
 94. The method of any of claims 91-93 comprisingcleaning the first reaction product before moving it to the secondchamber.
 95. The method of any of claims 91-93 further comprising, atleast once, moving a reaction product through a microfluidic channelinto a subsequent non-microfluidic chamber and performing a subsequentchemical reaction on the reaction product to create a subsequentreaction product.
 96. The method of any of claims 91-93 furthercomprising, at least once and before moving a reaction product into anon-microfluidic chamber, moving a reaction product through amicrofluidic channel into a microfluidic chamber and performing asubsequent chemical reaction on the reaction product to create asubsequent reaction product.
 97. A device comprising: a) a samplechannel having a channel inlet and a channel outlet; b) anelectrophoresis capillary having a capillary inlet and a capillaryoutlet, wherein the capillary comprises an electrically conductivemedium and is in communication with the sample channel at a point ofconnection; c) an anode and a cathode configured to apply a voltageacross the capillary inlet and capillary outlet, wherein one of theanode or cathode comprises a forked electrode wherein the forks are inelectrical communication with the sample channel on different sides ofthe point of connection; and d) a second electrode in electricalcommunication with the sample channel substantially opposite the pointof connection.
 98. The device of claim 97 wherein the second electrodeis comprised as a third fork in the forked electrode.